The three-dimensional trajectory data of vehicles have important practical meaning for traffic behavior analysis. To solve the problems of narrow visual angle in single-camera scenes and lack of continuous trajectories in 3D space by current cross-camera trajectory extraction methods, we propose an algorithm of vehicle spatial distribution and 3D trajectory extraction in this paper. First, a panoramic image of a road with spatial information is generated based on camera calibration, which is used to convert cross-camera perspectives into 3D physical space. Then, we choose YOLOv4 to obtain 2D bounding boxes of vehicles in cross-camera scenes. Based on the above information, 3D bounding boxes around vehicles are built with geometric constraints which are used to obtain projection centroids of vehicles. Finally, by calculating the spatial distribution of projection centroids in the panoramic image, 3D trajectories of vehicles are extracted. The experimental results indicate that our algorithm can effectively complete vehicle spatial distribution and 3D trajectory extraction in various traffic scenes, which outperforms other comparison algorithms. Vehicle spatial distribution and 3D trajectory extraction is an important sub-task in the field of computer vision. With the development of intelligent transportation systems (ITS), a large amount of vehicle trajectory data reflecting movements is obtained through traffic surveillance videos, which can be used for traffic behavior analysis ,2 such aIn current applications, trajectories mainly refer to two-dimensional trajectories in the image space, which do not contain spatial information of vehicles in the real world. Compared with 2D trajectories, 3D trajectories have one more dimension of spatial information, which has more obvious advantages in practical applications and can be further applied to traffic accident scene reconstruction and responsibility identification , as wellCurrently, the most commonly used methods for obtaining 3D vehicle trajectories are based on object detection and feature point methods ,12,13, wAt present, full space fusion mainly relies on multi-scene stitching methods, which can be divided into two categories. (1) Image stitching based on image alignment ,21. The Methods of vehicle trajectory extraction in the whole space is cross-camera vehicle tracking, which means obtaining continuous vehicle trajectory from images taken by multiple cameras with or without overlapping areas. These methods usually contain three essential steps: camera calibration, vehicle detection, and tracking in single-camera scenes and cross-camera vehicle matching. For cameras with overlapping areas, the spatial correlation can be calculated by overlapping areas to obtain continuous trajectories. However, in practical applications, “blind areas” are often existed in images taken by multiple cameras. In case of this condition, methods of re-identification are used to accurately and efficiently match vehicles in different perspectives through vehicle apparent features. Then, continuous vehicle trajectories in the whole space can be obtained by space-time information inference.Currently, re-identification methods used in cross-camera vehicle tracking are mostly based on vehicle features, such as vehicle color, shape, and texture, among which SIFT feature is the most commonly used due to its invariance to light, rotation, and scale. However, robustness of SIFT to affine transformation is low. To improve this problem, Hsu et al.  proposedA method of road space fusion in cross-camera scenes based on camera calibration is proposed to generate a road panoramic image with physical information, which is used to convert cross-camera perspectives into 3D physical space.A method of 3D vehicle detection based on geometric constraints is proposed to accurately obtain projection centroids of vehicles, which is used to describe vehicle spatial distribution in the panoramic image and 3D trajectory extraction of vehicles.In view of the problems existing in current cross-camera vehicle tracking methods, such as the influence of visual angle, vehicle occlusion, and lack of continuous trajectories in 3D space, we propose an algorithm of vehicle spatial distribution and 3D trajectory extraction in cross-camera traffic scene. The main contributions of this paper are summarized as follows:The rest of this paper is organized as follows. The proposed algorithm to complete vehicle spatial distribution and 3D trajectory extraction is illustrated in The overall flow chart of the proposed algorithm is shown in To complete road space fusion in cross-camera scenes, the relationship between 2D image space and 3D world space must be derived through camera calibration. In this paper, we refer to the study  and our Schematic diagram of coordinate system and camera calibration model is shown in In camera calibration, calibration parameters usually include camera focal length In this paper, the single vanishing point-based calibration method VWL ,32 is adAs shown in Besides vanishing points, markings on the road plane are also commonly used signs. In terature  that h cFrom Equation (6), To illustrate the road space in a straightforward way, the origin of the image coordinate system Then, the The translation matrix is:Therefore, the adjusted mapping from world point Finally, according to the derivation, the adjusted mapping between world and image can be described as follows:The mapping between world and image in a single scene can be described through camera calibration. To complete 3D vehicle trajectory extraction in cross-camera scenes, the road space needs to be fused. At present, image stitching methods are often used, but most of them rely on overlapping areas to extract feature points for matching and obtaining transformation of scenes. However, feature extraction and matching are time-consuming. For multi-scene (more than two scenes) stitching, accumulated errors are existed in transformation of scenes, which will affect the quality of final image stitching result and the measurement accuracy of physical distance. Therefore, we propose a road space fusion algorithm in cross-camera scenes based on camera calibration which is not completely dependent on overlapping areas between scenes. When there are no overlapping areas between scenes, only the distances between cameras are needed.Schematic diagram of road space fusion in cross-camera scenes is shown in Step 1:Camera calibration. The calibration method proposed in this paper is used to calculate calibration parameters of each camera in the scene, including internal parameter matrix Step 2:Road area identification by setting control points. Harris corner extraction algorithm is used to obtain the image coordinate set of the nearest and furthest marking endpoints on the road plane in each scene, which is denoted as Step 3:Set control parameter groups and divide pixels of the panoramic image Step 4:Generate the complete panoramic image Schematic diagram of road distribution in the panoramic image is shown in The pixel in the road area panoramic image-to-worldSub-scene world-to-panoramic imageSince the generated panoramic image contains physical information of road space, the position in the sub-scene world coordinate system and the unified world coordinate system can be calculated directly from a point in the panoramic image. In addition, the position in the unified world coordinate system and the panoramic image coordinate system can also be analyzed from a point in the sub-scene world coordinate system. The specific mapping equation group is as follows:Based on road space fusion in cross-camera scenes, to further obtain vehicle spatial distribution and 3D trajectory, vehicle detection in the scene is needed. Since the height of vehicle feature points is unknown, projection centroid is adopted in this paper instead, which depends on 3D vehicle detection. Considering actual application requirements, we choose YOLOv4  for 2D vStep 1:YOLOv4 is used to obtain the vertices in the image coordinate system Step 2:Suppose 3D vehicle physical size Step 3:The calculation results in Step 2 are converted to the image coordinates Schematic diagram of 2D/3D vehicle detection is shown in According to the above 3D vehicle detection algorithm, obtaining accurate 3D vehicle physical size is the premise to complete precise 3D vehicle detection. Due to the factor of perspective distortion and lack of depth information in monocular image, accurate size cannot be obtained by vehicle type which is derived from YOLOv4. Therefore, geometric constraints are considered to accurately calculate 3D vehicle physical size, which includes diagonal constraint and vanishing point constraint.3D vehicle detection is equivalent to obtaining 3D vehicle physical size According to 3D bounding box model, The difference of Equation (11) and (12) consists of a set of diagonal constraint. According to the principle of vanishing point, the straight line composed of 0–3, 1–2, 4–7, 5–6 point pairs in the 3D bounding box model must pass the vanishing point along the road direction in the image coordinate system. Therefore, it can be used as another set of constraints to accurately calculate 3D vehicle physical size.In the image coordinate system, the included angle between two lines  can be denoted as For four point pairs, according to the cosine theorem, we can derive The sum of four equations above consists of a set of vanishing point constraint. As shown in Step 1:YOLOv4 is used to obtain the vertices in the image coordinate system Step 2:Step 3:According to Equation (11), the diagonal pixel length of 2D bounding box is calculated. Then, the world coordinates of vertex 1 and 7 are converted to the image coordinates according to Equation (7) as Step 4:According to Equation group (8), the world coordinates of vertices from 0 to 7 are converted to image coordinates as In this paper, the steps to obtain the vehicle geometric constraints are as follows:According to the above algorithm, the diagonal constraint and vanishing point constraint are obtained to construct the constraint error as To sum up, the nonlinear constraint function of the parameter The constraint function is nonlinear. LM (Levenberg-Marquardt) method is adopted in this paper to solve the constraint function, which is easy to reach convergence. The initial value standard  based onAfter solving accurate 3D vehicle physical size, 3D vehicle detection can be completed. Then, the world coordinates of projection centroids can be calculated. According to Equations (9) and (10), coordinates of vehicles in the panoramic image and other scenes can be obtained. As shown in In our experiments, we used the Intel Core i7-8700 CPU, NVIDIA 1080Ti GPU (Graphics Processing Unit), 32GB memory, and Windows 10 operating system. The open source framework Darknet is used for vehicle detection.Experiments are carried out on the public dataset BrnoCompSpeed  and actuDue to the lack of cross-camera datasets from road surveillance perspectives, we choose a public dataset of single-camera scenes from surveillance perspectives published by researchers of Brno University of Technology for our experiments. The cross-camera dataset made by ourselves and experiments carried out on this scene are described in detail in The public dataset BrnoCompSpeed contains six traffic scenes captured by roadside surveillance cameras. Each scene can be divided into left, middle, and right perspectives, with a total of 18 HD (High Definition) videos (about 200 GB). The resolution of all the videos is 1920 × 1080. The dataset contains various types of vehicles such as hatch-back, sedan, SUV, truck and bus, and the position and velocity of vehicles are accurately recorded by radar. Therefore, this dataset can be used to verify the accuracy of vehicle spatial distribution and 3D trajectories in single-camera scenes.As shown in The real position of the vehicle in the world coordinate system is defined as Examples of the vehicle spatial distribution and 3D trajectories in dataset scenes are shown in From the experimental results, it can be seen that the average error of vehicle spatial distribution within the scope of hundred meters is less than 5%, which means the accuracy can reach the centimeter level. In the meanwhile, the proposed algorithm is also adaptable to the situation of part vehicle occlusion.To further verify the application ability of the proposed algorithm, we choose the actual road with large traffic flow which is located on the Middle Section of South Second Ring Road in Xi’an, ShaanXi Province, China to make a small dataset of cross-camera scenes. The dataset consists of three groups of HD videos , and each of which is about 0.5 h long. The resolution of all the videos is 1280 × 720. In our experiment, we choose three examples of vehicles, which are shown in As shown in Since the proposed 3D vehicle detection algorithm is based on geometric constraints, the overall processing speed is fast. It can be seen from examples in During the experiment, it can also be found that the accuracy of vehicle spatial distribution and 3D trajectory is related to the pan angle Through experimental verification, the proposed algorithm of vehicle spatial distribution and 3D trajectory extraction in cross-camera scenes in this paper has achieved good results in both BrnoCompSpeed dataset single-camera scenes and actual road cross-camera scenes. The main contributions of this paper are as follows: (1) A road space fusion algorithm in cross-camera scenes based on camera calibration is proposed to generate the panoramic image with physical information in road space, which can be used to convert multiple cross-camera perspectives into continuous 3D physical space. (2) A 3D vehicle detection algorithm based on geometric constraints is proposed to accurately obtain 3D vehicle projection centroids, which is used to describe vehicle spatial distribution in the panoramic image and to extract 3D trajectories. Compared with existing vehicle tracking methods, continuous 3D trajectories can be obtained in the panoramic image with physical information by 3D projection centroids, which is helpful to applications in large scope road scenes.However, 3D vehicle projection centroids obtained by the proposed algorithm in this paper is highly dependent on 2D vehicle detection results. When the vehicle is far from the camera, it is prone to be missed of detection and the accuracy will decrease when the camera pan angle is close to 0°. Moreover, the proposed algorithm cannot currently be adapted to various road situations and congested traffic. In future work, a more efficient method for road space fusion can be developed to generate the panoramic image and calculate vehicle spatial distribution more precisely and a more sophisticated vehicle detection network can be designed to fuse various types of geometric constraints to further improve the accuracy of 3D vehicle detection under different camera pan angles. In addition, only straight roads and simple traffic conditions are considered in this paper, which is necessary to be further extended to complex traffic scenes such as road-crossing (containing winding roads) and traffic congestion for more practical and advanced applications. Efforts are also needed to collect a large dataset of these complex traffic scenes for algorithm validation. This direction is a key and difficult point in the future work.


The progressive patterns of neurological disability in multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD) and the significance of clinical relapses to the progressions of neurological disability in these diseases have not been fully elucidated. In this study, to elucidate the impact of relapses to the progression of accumulated neurological disability and to identify the factors to affect the progression of neurological disability in MS and NMOSD, we followed 62 consecutive MS patients and 33 consecutive NMOSD patients for more than 5 years with the clinical symptoms, relapse occurrence, and Expanded Disability Status Scale (EDSS) in the chronic phase. All enrolled MS patients were confirmed to be negative for serum anti-myelin oligodendrocyte glycoprotein antibody. As a result, patients with NMOSD showed significantly severer neurological disability at 5 years from onset than MS patients. Progression in EDSS score was almost exclusively seen after clinical attacks in NMOSD, whereas progression could be observed apart from relapses in MS. Neurological disability did not change without attacks in NMOSD, whereas it sometimes spontaneously improved or deteriorated apart from relapses in MS (p < 0.001). In patients with MS, those with responsible lesions primarily in spinal cord were more likely to show such spontaneous improvement. In conclusion, clinical deterioration in NMOSD patients is irreversible and almost exclusively takes place at the timing of clinical attacks with stepwise accumulation of neurological disability. Meanwhile, changes in EDSS score can be seen apart from relapses in MS patients. Neurological disability in MS patients is partly reversible, and the patients with disease modifying drugs sometimes present spontaneous improvement of the neurological disability. Both diseases typically present recurrent clinical attacks with lesions in cerebrum, optic nerves, brainstem, and spinal cord3. At present, MS is diagnosed based on the dissemination of lesions in time and space6, whereas NMOSD is diagnosed both by the clinical history and the presence of serum anti-aquaporin-4 antibody (AQP4-IgG)7.Multiple sclerosis (MS) and neuromyelitis optica spectrum disorders (NMOSD) are major autoimmune-related neurological diseases that predominantly impairs the central nervous system (CNS) but have distinct pathophysiological mechanisms8. Based on this concept, clinical course of MS is generally categorized into the following three subtypes: primary progressive MS (PPMS), relapsing–remitting MS (RRMS), and secondary progressive MS (SPMS)10. Meanwhile, progressive pattern of neurological impairment in NMOSD has not been so much studied until now, although the subsequent neurological disability is generally severer in NMOSD than in MS13. From before, the progression of neurological disability in NMOSD has been empirically thought to occur mainly at the timing of clinical attacks14, but whether it occurs even during the intermittent period between attacks or not has not been proved yet. Because the expected clinical course will surely affect the achieved outcomes in clinical studies enrolling the patients with these diseases, elucidating the characteristic clinical course and factors that affect the progression of neurological disability in these diseases has many clinical significances.The accumulation of neurological impairment in MS is thought to be mainly comprised of the following two components: subclinical progressive brain atrophy and recurrent clinical relapses during which the responsible lesions are contrast-enhanced in MRIIn this study, we enrolled an enough number of MS and NMOSD patients who have their clinical onset between 2000 and 2015, and followed their neurological disability every year from the first visit to our hospital. The progressive pattern of their neurological disability was evaluated with other information such as patient background and relapses to identify the clinical factors that mainly regulate the progression of neurological disability in each of MS and NMOSD.15. The presence of serum AQP4-IgG in the enrolled NMOSD patients was also confirmed based on the cell-based assay method17.For this study, a total of 62 consecutive MS patients and 33 consecutive AQP4-IgG-positive NMOSD patients, who had their clinical onset between 2000 and 2015 and treated in our university hospital were initially collected. These initial cohorts were prospectively followed up, among which 57 MS patients (91.9%) and 31 NMOSD patients (93.9%) were followed and treated in our university hospital for more than 5 years from their onset. All enrolled MS patients were confirmed to be negative for the presence of serum anti-myelin oligodendrocyte glycoprotein (MOG) antibody by utilizing the cell-based assay methodAll of the 5 MS patients who was not followed for more than 5 years dropped out because of moving. One of the 2 NMOSD patients who was not followed for more than 5 years died because of a malignant tumor 2 years after the onset of NMOSD, but another patient dropped out because of moving.18. Because neurological disability can drastically exacerbate and recover in the acute and subacute phases of clinical relapses with proper treatments with immune-suppressants, EDSS scores within 3 months from the last relapse were not used in this study. Relapses in MS and NMOSD were defined by the presence of clinically evidenced gadolinium-enhanced T1-weighted lesions with the neurological symptoms sustained for more than 24 h in the absence of fever or infection4.In these patients, comprehensive clinical information was collected, such as onset age, sex, follow up period as of 2019, types of relapse preventive therapies, type and timing of clinical attacks, and the titer of serum anti-AQP4 antibody. The irreversible neurological impairment was evaluated with the Expanded Disability Status Scale (EDSS) in every 1 or 2 years in these patients19.As for other clinical information, data about the number of cerebral lesions at 5 years from the onset and the site of lesion responsible for the neurological disability  were also collected in MS patients. To identify the factors that affect the neurological disability in MS, we also evaluated the gray matter volume and white matter lesion volume in some of them with a volumetric method as previously reportedComparisons of two distributions were performed with either of the Student’s t-test or Mann–Whitney U test, based on the normality of distributions. Comparisons of frequencies were performed with either of the chi-squared test or Fisher’s exact test, based on the size in each cell. A value of p < 0.05 was regarded to be statistically significant. The analyses were conducted using either SPSS Statistics Base 22 software  or MATLAB R2015a .This study was approved by the institutional review board of the Tohoku University Graduate School of Medicine , and was carried out in accordance with the standards stated in the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.Informed consent was obtained from all individual participants included in the study.The total follow-up period with EDSS evaluation in the initially collected MS patients with onset between 2000 and 2015 was 337 person-year (62 patients) as of Oct 2019; that in the initially collected NMOSD patients with onset between 2000 and 2015 was 229 person-year (33 patients). Patients’ background in both groups are listed in Table The progressions of EDSS in both disease groups by years from the onset, irrespective of the length of follow-up period or the occurrence of relapses, are shown in Fig. Within the initially enrolled patients, 53 MS patients were evaluated with EDSS at 5 years from the onset and 20 MS patients were evaluated at 10 years from the onset. In NMOSD group, all 31 patients were evaluated with EDSS at 5 years from the onset and 18 patients were evaluated at 10 years from the onset. The distributions of EDSS in MS and NMOSD groups at 5 and 10 years from the onset are listed in the middle of Table Data regarding to the relationship between attacks and the progression of irreversible neurological disability, irrespective of relapses, are summarized in the lower half of Table When focusing on the period without clinical relapses, 255 (85.3%) of the 299 person-years of follow-up in MS group showed unchanged EDSS score, whereas 197 (95.6%) of the 206 follow-up years in NMOSD group showed unchanged EDSS score . In other words, neurological disability in NMOSD hardly changes without clinical attacks, whereas that in MS is more likely to change without relapses.To visually confirm the difference in the impact of relapse to neurological disability between MS and NMOSD, we depicted line graphs of chronological change in EDSS for each patient by the relapse occurrence as shown in Fig. As can be seen in the figures, not a few MS patients with proper treatments with DMDs showed a spontaneous EDSS improvement during their clinical course irrespective of relapse occurrence. The frequency of EDSS annual improvement in MS and NMOSD patients is shown in the bottom of Table 20. As a result, there were 12 patients whose EDSS improved with clinically important difference in the first 5 years from the onset, and 7 patient whose EDSS deteriorated with clinically important difference in the first 5 years from the onset. The characteristics of these two groups are summarized in Table In MS group, the prevalence of minimally clinically important difference in EDSS score in the first 5 years was evaluated. Based on a previous literature, minimal clinically important difference was defined as 1.0 point change when the EDSS score was 0–5.0, and as 0.5 point change when the EDSS score was 5.5–8.522. More importantly, EDSS in MS patients with DMDs often showed spontaneous sustainable improvement, which was hardly observed in the NMOSD patients. Such spontaneous EDSS improvement was likely to be seen in MS patients with neurological impairment based on myelitis and with less cerebral lesions.Based on the results of this study, progressive pattern of neurological disability in MS and NMOSD was suggested to be largely different. Progression of EDSS in NMOSD mainly took place at the timing of each attack occurrence, and it did not deteriorate for long time if there is no attack occurrence. Meanwhile, progression of EDSS in MS mainly took place irrespective of the relapse occurrence as previously known25. It may be better in upcoming clinical trials to distinguish the MS patients with only a few cerebral lesions, who are expected not to show EDSS deterioration in the following several years, and those with much more cerebral lesions, who are likely to show EDSS deterioration in the following several years.One notable finding of this study was that neurological impairment in MS could be spontaneously improved, suggesting that damages in oligodendrocytes could be partially reversible or replaceable. Such spontaneous improvement was mainly seen in MS patients with responsible lesions in the spinal cord, whereas the MS patients with responsible lesions in the cerebrum or brainstem did not show such spontaneous remission. It is widely known that MS patients with more cerebral lesions and larger cerebral lesions are likely to show accelerated neurological disability and faster rate of global brain atrophy26. Also, this study indicated that one of the most appropriate primary outcomes to be evaluated in trials with NMOSD patients is the subsequent attack frequency or the severity of neurological damage in each attack, as neurological disability in NMOSD shows stepwise accumulation as a result of attacks27. Meanwhile, in clinical trials in MS, relapse frequency would not be among the most important enrollment criteria or outcomes to be evaluated, because EDSS improvement and deterioration often take place irrespective of the relapse occurrence. Suppression of neurological disability progression irrespective of relapses or facilitation of spontaneous EDSS improvement would be more desirable therapeutic outcome in MS patients. Since not a few MS patients showed spontaneous improvement in neurological disability, the achievement rate of EDSS recovery may be another possible outcome to be evaluated in the future clinical trials in MS.The absence of spontaneous change in EDSS score independent from attacks in NMOSD patients supports the importance of taking the preceding relapse frequency in the last several years into consideration at the enrollment of clinical trials with NMOSD patients28. Lastly, we did not study EDSS progression in patients with serum anti-MOG antibody, which is expected to be elucidated in the future researches.There are several limitations in this study. First, almost all of the enrolled patients were Asian, except for one Caucasian female MS patient. Thus, whether the suggested spontaneous EDSS improvement in MS patients with DMDs can be also observed in MS patients of Caucasians and African-Americans is to be elucidated in the future clinical researches. Second, almost all of the enrolled patients were treated with standard relapse preventive therapies. Thus, the natural progressive patterns of neurological disability without any treatments are not known. Moreover, most of the studied MS patients in this study changed DMDs from IFN-β to fingolimod during the follow-up period. Thus, whether different types of DMDs may produce different rates of spontaneous remission in MS patients or not is unconcluded. However, such possibility seems to be unlikely between IFN-β and fingolimod, as there was no significant difference in the progression of neurological disability between these two drugs in a previous randomized double-blind studyNMOSD patients with relapse-preventive therapies show EDSS progression exclusively at the timing of each clinical attack, and EDSS does not deteriorate without attacks, suggesting that preventing clinical attacks is the most important therapeutic target in NMOSD. Meanwhile, MS patients treated with DMDs often show spontaneous improvement or deterioration in EDSS independent from relapses. Because the neurological disability in MS is reversible to some extent, therapeutic strategies to facilitate such spontaneous improvement in MS would be a promising therapeutic strategy in the future.


We propose that, by providing Atp9 for biogenesis of ATP synthase, Atco complexes free Cox6 for assembly of COX. This suggests that Atco complexes may play a role in coordinating assembly and maintaining proper stoichiometry of the two oxphos enzymesMitochondrial oxidative phosphorylation (oxphos) is the process by which the ATP synthase conserves the energy released during the oxidation of different nutrients as ATP. The yeast ATP synthase consists of three assembly modules, one of which is a ring consisting of 10 copies of the Atp9 subunit. We previously reported the existence in yeast mitochondria of high molecular weight complexes composed of mitochondrially encoded Atp9 and of Cox6, an imported structural subunit of cytochrome oxidase (COX). Pulse-chase experiments indicated a correlation between the loss of newly translated Atp9 complexed to Cox6 and an increase of newly formed Atp9 ring, but did not exclude the possibility of an alternate source of Atp9 for ring formation. Here we have extended studies on the functions and structure of this complex, referred to as Atco. We show that Atco is the exclusive source of Atp9 for the ATP synthase assembly. Pulse-chase experiments show that newly translated Atp9, present in Atco, is converted to a ring, which is incorporated into the ATP synthase with kinetics characteristic of a precursor-product relationship. Even though Atco does not contain the ring form of Atp9, cross-linking experiments indicate that it is oligomeric and that the inter-subunit interactions are similar to those of the  In mammalian mitochondria the NADH-coenzyme Q reductase, the bc1 and cytochrome oxidase (COX) complexes are physically associated in a supercomplex or respirasome Reviewers' comments:Reviewer's Responses to QuestionsComments to the Author1. Is the manuscript technically sound, and do the data support the conclusions?The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: YesReviewer #2: Yes**********2. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: I Don't KnowReviewer #2: Yes**********3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.The Reviewer #1: YesReviewer #2: Yes**********4. Is the manuscript presented in an intelligible fashion and written in standard English?PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.Reviewer #1: YesReviewer #2: Yes**********5. Review Comments to the AuthorPlease use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: This manuscript by the Tzagoloff’s group builds on their previously reported observation that in yeast mitochondria, newly-synthesized Atp9, a mitochondrion encoded ATPase subunit, forms high molecular weight complexes with Cox6, a nucleus encoded subunit of cytochrome c oxidase. These complexes, now called ATCO, are proposed to coordinate the assembly of the two oxphos complexes.The experiments presented clearly demonstrate that the ATCO complexes are the source of Atp9 for Atp9 ring formation, are well executed and grant publication. However, the fact that in the absence of Cox6, Atp9 assembly proceeds normally, makes the model of co-assembly regulation contra intuitive. Unfortunately, the authors did not explore here whether ATCO is aprecursor of COX and the sole source of Cox6 for COX assembly. Is has been previously reported by several groups that defects in mitochondrial ATPase biogenesis lead to a COX assembly defect, but not vice versa. Could this be related to the unavailability of Cox6 for COX assembly? The authors should at least discuss these possibilities.Minor points:1- There are a few typos/grammar issues to be fixed. For example- Line 89: “nuclear encoded” should be “nucleus-encoded”.- Line 169: “does not translated Cox1” should be “does not translate Cox1”Reviewer #2: The authors previously reported the existence of high molecular weight complexes (referred to as ATCO) containing the mtDNA-encoded Cox6 and Atp9 proteins. The present study shows that newly synthesized Atp9 associates with ATCO before being incorporated into ATP synthase as an oligomeric ring. The use of cysteine atp9 mutants provides solid evidence for Atp9-Atp9 interactions in ATCO similar to those in assembled ATP synthase, arguing against non-specific aggregation of Atp9 and Cox6. Based on these findings, the authors propose that ATCO may regulate the relative amounts of COX and ATP synthase in being a source of Cox6 and Atp9 to be incorporated synchronously into their cognate complexes. This is an important and well executed study that helps to better understand the mechanisms involved in formation of the mitochondrial energy-transduction system.Minor points.- A comment on ‘the stand-alone Atp9-ring would be appreciated. Does its detection indicate that the Atp9-ring forms from ATCO independently of any other ATP synthase component, which is suggested by the model. How then the ring is produced in stoichiometric amounts with the other ATP synthase modules? Could the authors evoke a possible mechanism?-As nicely discussed, COX is much more sensitive to glucose repression than ATP synthase is, which provides evidence that Cox3 is not essential for formation of the Atp9-ring and its incorporation into ATP synthase. The converse is seemingly not true. Indeed, yeast ATP synthase defective mutants show a decreased content in COX, except those with FO-mediated proton leaks. It has been argued recently :3792-3804, and references therein) that the proton-translocation activity of ATP synthase modulates the rate of COX biogenesis through the mitochondrial membrane electrical potential. Although this is not a requirement, this reviewer would appreciate if the authors could comment these reported findings in the light of the present study.-In Fig. 6B, RKY28 should be replaced by RKY26.**********what does this mean?). If published, this will include your full peer review and any attached files.6. PLOS authors have the option to publish the peer review history of their article  digital diagnostic tool,  28 Apr 2020Dear Editor,Thank you for the prompt review of our manuscript “ATCO, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase ”. We also thank the two reviewers for their thoughtful comments and suggestions, which we have tried to incorporate into the revised manuscript. The changes made are summarized below. Reviewer #1: This manuscript by the Tzagoloff’s group builds on their previously reported observation that in yeast mitochondria, newly-synthesized Atp9, a mitochondrion encoded ATPase subunit, forms high molecular weight complexes with Cox6, a nucleus encoded subunit of cytochrome c oxidase. These complexes, now called ATCO, are proposed to coordinate the assembly of the two oxphos complexes.The experiments presented clearly demonstrate that the ATCO complexes are the source of Atp9 for Atp9 ring formation, are well executed and grant publication. However, the fact that in the absence of Cox6, Atp9 assembly proceeds normally, makes the model of co-assembly regulation contra intuitive. Unfortunately, the authors did not explore here whether ATCO is aprecursor of COX and the sole source of Cox6 for COX assembly. Is has been previously reported by several groups that defects in mitochondrial ATPase biogenesis lead to a COX assembly defect, but not vice versa. Could this be related to the unavailability of Cox6 for COX assembly? The authors should at least discuss these possibilities.The question of whether Cox6 of ATCO is a precursor for COX biogenesis is being actively studied in our lab but has yet to be confirmed or refuted. At present we have evidence that the pleiotropic deficiency of cytochrome oxidase in mitochondria of ATP synthase mutants is related to decreased translation of all three mitochondrially encoded subunits of this respiratory complex. We do not know if Cox6 is involved in this translational regulation. This will require overcoming some sticky technical problems that we are working on now. The discussion of the effect of ATP synthase mutations on cytochrome oxidase has been expanded. Our recent experiments show that growth of an atp9 null mutant under conditions that produce only 50% �-/0 cells result in a 10 fold reduction of Cox6 (unpublished). The explanation for this could that the absence of Atp9 leads to a decreased membrane potential that prevents Cox6 transport (see response last question). Alternatively, transport of Cox6 is normal but in absence of Atp9, it is rapidly proteolyzed. In both cases Cox6 would be unavailable for COX biogenesis.Reviewer #2: The authors previously reported the existence of high molecular weight complexes (referred to as ATCO) containing the mtDNA-encoded Cox6 and Atp9 proteins. The present study shows that newly synthesized Atp9 associates with ATCO before being incorporated into ATP synthase as an oligomeric ring. The use of cysteine atp9 mutants provides solid evidence for Atp9-Atp9 interactions in ATCO similar to those in assembled ATP synthase, arguing against non-specific aggregation of Atp9 and Cox6. Based on these findings, the authors propose that ATCO may regulate the relative amounts of COX and ATP synthase in being a source of Cox6 and Atp9 to be incorporated synchronously into their cognate complexes. This is an important and well executed study that helps to better understand the mechanisms involved in formation of the mitochondrial energy-transduction system.- A comment on ‘the stand-alone Atp9-ring would be appreciated. Does its detection indicate that the Atp9-ring forms from ATCO independently of any other ATP synthase component, which is suggested by the model. How then the ring is produced in stoichiometric amounts with the other ATP synthase modules? Could the authors evoke a possible mechanism?The ATP9 ring by itself has also been reported to be present in an atp6 mutant, a mutant with an unprocessed N-terminal presequence on Atp6 and an atp10 mutant that contains only 5% of the normal concentration of ATP synthase (unpublished). Although these observations indicated that Atp9 can oligomerize into the ring independent of the assembly of the rest of the complex, they do not exclude the possibility that At9 translation and/or ring formation is regulated as the amount of ring formed in these studies was not measured. Mitochondria from cells that have been incubated for two hour growth in the presence of chloramphenicol have a significantly higher concentration of ATCO. The incubation in the presence of chloramphenicol inhibits mitochondrial translation of Atp9 but not of Cox6, which would be expected to increase the mitochondrial pool of this subunit and hence also increase the amount of ATCO when Atp9 translation is restored in isolated mitochondria. This may indicated that Atp9 translation and ATCO formation may be regulated by Cox6. This is also supported by previous evidence that cells undergoing derepression from glucose assemble significantly less Atp9 ring in a cox6 but not other oxidase mutants . This is now mentioned in the discussion.-As nicely discussed, COX is much more sensitive to glucose repression than ATP synthase is, which provides evidence that Cox3 is not essential for formation of the Atp9-ring and its incorporation into ATP synthase. The converse is seemingly not true. Indeed, yeast ATP synthase defective mutants show a decreased content in COX, except those with FO-mediated proton leaks. It has been argued recently :3792-3804, and references therein) that the proton-translocation activity of ATP synthase modulates the rate of COX biogenesis through the mitochondrial membrane electrical potential. Although this is not a requirement, this reviewer would appreciate if the authors could comment these reported findings in the light of the present study.Su et al (2019) have shown that overexpression of the oxodicarboxylate carrier OCD1 an atp6 mutant increases the concentrations of ATP synthase and COX a factor 2.5 and 4, respectively. These authors interpreted their results to indicate the higher abundance of COX enhances substrate-level ATP synthesis as a result of increased TCA cycle flux, thereby restoring the membrane potential of mitochondria in the atp6 mutant. Since most of the ATP in the ODC1/atp6 mutant is made by substrate level phosphorylation during conversion of α-ketoglutarate to succinate Su et al (2019) proposed that COX biogenesis is modulated by the membrane potential rather than by ATP from oxidative phosphorylation.The proposed function of ATCO in establishing the stoichiometry ATP synthase relative to cytochrome oxidase is predicated on the absence in normal cells of monomeric Atp9 and Cox6. This is supported by our results in the case Atp9. Although we do not have similar direct evidence for Cox6, the observed increase synthesis of ATCO by isolated mitochondria from cells that have been grown in the presence of chlroramphenicol, support the idea that synthesis of Atp9 and more to the point also of ATCO depends on the supply of Cox6. This point is now brought out in the discussion. Like other proteins that are transported by the TIM23 translocase of mitochondria, uptake of Cox6 depends on a membrane potential. The restoration of a membrane potential in the atp6 mutant overexpressing ODC1 strain would be expected to improve transport of newly synthesized Cox6 from the cytoplasm to the matrix, which in turn could account for the increase of COX observed by Su et al (2019). The typos and other minor points picked up the reviewers have been corrected.We hope that with these improvements the paper will be deemed acceptable for publication.Sincerely yours,Alex TzagoloffAttachmentresponse to reviewers.docxSubmitted filename: Click here for additional data file. 30 Apr 2020ATCO, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthasePONE-D-20-04750R1Dear Dr. Tzagoloff,We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.Within one week, you will receive an e-mail containing information on the amendments required prior to publication. 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Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact With kind regards,Yidong BaiAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 4 May 2020PONE-D-20-04750R1 Atco, a yeast mitochondrial complex of Atp9 and Cox6, is an assembly intermediate of the ATP synthase Dear Dr. Tzagoloff:I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please notify them about your upcoming paper at this point, to enable them to help maximize its impact. If they will be preparing press materials for this manuscript, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. 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Even when resting pulse oximetry is normal in the patient with acute Covid-19, hypoxia can manifest on exertion. We summarise the literature on the performance of different rapid tests for exertional desaturation and draw on this evidence base to provide guidance in the context of acute Covid-19.What exercise tests have been used to assess exertional hypoxia at home or in an ambulatory setting in the context of Covid-19 and to what extent have they been validated?What exercise tests have been used to assess exertional hypoxia in other lung conditions, to what extent have they been validated and what is the applicability of these studies to acute Covid-19?AMED, CINAHL, EMBASE MEDLINE, Cochrane and PubMed using LitCovid, Scholar and Google databases were searched to September 2020. Studies where participants had Covid-19 or another lung disease and underwent any form of exercise test which was compared to a reference standard were eligible. Risk of bias was assessed using QUADAS 2. A protocol for the review was published on the Medrxiv database.Of 47 relevant papers, 15 were empirical studies, of which 11 described an attempt to validate one or more exercise desaturation tests in lung diseases other than Covid-19. In all but one of these, methodological quality was poor or impossible to fully assess. None had been designed as a formal validation study (most used simple tests of correlation). Only one validation study  contained sufficient raw data for us to calculate the sensitivity (88%), specificity (81%) and positive and negative predictive value (79% and 89% respectively) of the 1MSTST. The other 4 empirical studies included two predictive studies on patients with Covid-19, and two on HIV-positive patients with suspected pneumocystis pneumonia. We found no studies on the 40-step walk test . Heterogeneity of study design precluded meta-analysis.Exertional desaturation tests have not yet been validated in patients with (or suspected of having) Covid-19. A stronger evidence base exists for the diagnostic accuracy of the 1MSTST in chronic long-term pulmonary disease; the relative intensity of this test may raise safety concerns in remote consultations or unstable patients. The less strenuous 40-step walk test should be urgently evaluated.The online version contains supplementary material available at 10.1186/s13643-021-01620-w. A substantial proportion of patients with acute coronavirus 19 (Covid-19) develop a potentially critical form of the illness requiring intensive care unit admission . PatientThe poor correlation between both subjective feeling of shortness of breath (dyspnoea) and objective measures of breathlessness and hypoxia in patients with Covid-19 has resulted in UK guidelines recommending that the assessment of the breathless, unwell or high-risk patient should include oximetry . For exaIndeed, the mismatch between relatively mild subjective respiratory distress and objective evidence of peripheral hypoxia is now recognised as a feature of Covid-19 and has been termed ‘silent’ or ‘happy’ hypoxemia , 12. Sim2/FiO2 ratios and requirement for supplemental oxygen in 41%  this term only#3 (coronavirus OR covid*):ti,ab,kw#4 #2 or #3#5 #1 AND #4Search of Cochrane Library—Issue 10 of 12, October 2020:‘step test’ OR ‘walk test’ OR ‘field test’ OR ‘chair rise’ OR ‘sit to stand’ OR ‘exercise test’ OR ‘exercise testing’Search of Cochrane COVID-19 study register:Target condition: Individuals with suspected Covid-19 or another lung disease with or without symptoms.Index test: Any form of rapid exercise test performed at home or in a healthcare setting.Reference standards: 6MWT or CPET (which are the most commonly used in exercise testing), or a diagnostic test to diagnose the disease in question such as bronchoalveolar lavage to diagnose pneumocystis pneumonia.Study designs: clinical practice guidelines and systematic reviews addressing desaturation in exercise tests in lung disease, using the Cochrane definition of a Systematic Review  was demanding for people with ILD [interstitial lung disease]’ (page 15).In a further small study of 15 participants with pulmonary fibrosis, Labrecque et al. compared the 1MSTST (done twice to assess reproducibility), 6MWT and CPET . The maiWe placed less weight on the final five studies—as the methodological quality was uncertain, or because methodological quality was considered to be poor on our risk of bias and applicability tool, though the findings from all these additional studies were similar to the above ones. These studies were Gruet et al.  ; Morita The test that is most used in acute practice is the 40-step walk test—the patient is asked to walk 40 steps on the flat and oximetry repeated. We found no research studies on this at all.This rapid review has produced several key findings relevant to the assessment of exertional desaturation in patients with suspected Covid-19. First, we identified no published studies which had compared the performance of different brief exercise tests in a cohort of patients with (or suspected of having) Covid-19.correlated the level of exertional desaturation on a range of exercise tests with one or both accepted gold standard tests (6MWT and CPET) in various acute or chronic lung conditions, none of these studies had been designed as a formal diagnostic test validation study. Rather, the focus had been on comparing the average SpO2 in the same group of patients who underwent both tests and showing no statistical difference between the two measurements. It is reassuring that there was high correlation between the 6MWT or CPET and the shorter 1MSTST, but we would expect the results of these tests to be correlated . Correlation alone, however, does not validate the test [Second, in all but one of 11 studies presented as ‘validation studies’ of brief exercise tests for assessing exertional desaturation in other lung diseases, methodological quality was poor or impossible to fully assess. Furthermore, whilst the authors of all 11 studies had the test .Only one of the 11 studies  contain2 levels even after the test had been completed [The third key finding of this review is that the 1MSTST produced a high cardiorespiratory stress , and patients with lung disease continued to show a further drop in SpOompleted , 52. In Finally, we identified a significant gap in the literature, namely the lack of validation studies (or indeed any relevant research) on the less strenuous 40-step test, which features in local and national guidance for Covid-19  and is i2 , rather than oxygen saturation per se. Given Covid-19’s physiological affinity with other perfusion-defect lung conditions such as interstitial lung disease and pneumocystis pneumonia, it follows that the assessment of exertional desaturation would be useful.As the studies listed in Table As none of the exertional tests have yet been validated to show that assessing exertional desaturation is better than routine clinical assessment at demonstrating increased risk, their use remains pragmatic. The resulting evidence gap in this regard will most likely be addressed in the near future given the continuing incidence of Covid-19.Our review findings, combined with what is known about the pathophysiology of acute Covid-19 , 13, 37,Current evidence therefore supports the recommendation to undertake a thorough clinical assessment and evaluation including a history, assessment of risk factors, pulse, respiratory rate and oxygen saturations. There is a physiological basis for adding graded assessment into the process of clinical evaluation as a positive test would raise significance.The ‘40 steps around the room test’ or its alternative ‘40 steps on the spot test’ (in a patient able to stand safely unaided and whose resting saturation is 96% or above) is the lowest level of exertion of any test either in the literature or in clinical practice. An alternative would be the ‘40 steps on the spot test’ with the patient standing in front of a chair in case of needing to sit down. This is appropriate for the home environment (no clinician on hand to guide or resuscitate) given the high-risk group. This test will likely have a low sensitivity but using basic physiological principles, if positive it is likely to be highly specific and warrant urgent assessment.If the 1MSTS is used, it should be followed by monitoring for at least one minute to observe for desaturation. Indeed, a short test means that it can take time to utilise the free oxygen in the blood stream and therefore witness the impact of desaturation. Such a test should only be done with another person  in attendance or nearby. The patient should be kept on continuous oximetry monitoring for at least 1 min following the test; the 1MSTST should never be attempted with a patient home alone.Our recommendation with regards to oxygen saturation monitoring would therefore be to measure baseline saturations, followed by the 40-step test, followed by the 1 MSTST . If oxygen saturations show a decrease at any of these stages, then the next step should not be attempted.Oximeters in smartphone apps are unreliable , so an aRisk to the patient from exercise tests should be considered. Patients should be advised to terminate promptly if they develop any adverse symptoms  . Tests inot advised to complete ‘as many as you can’ in the 1MSTST). We hypothesise that they are specific but not sensitive , though there is currently no hard data on this.The 40-step test  and the 1MSTST are considered the least demanding tests and therefore may be the most appropriate for recommending to patients at home , in the context of Covid-19. Furthermore, an understanding of how best to ask the patient about breathlessness on exertion, and how this correlates with exertion oximetry, could also help in the assessment of hypoxia in Covid-19.Finally, it is important to remember that a pulse oximeter reading, whether at rest or in an exertional test, does not replace thorough clinical assessment. A normal test does not necessarily mean the patient can be reassured that all is fine. Nevertheless, exertional hypoxia of 3% or more is a significant finding and always warrants further assessment and investigation.Additional file 1.Additional file 2.Additional file 3.


In late December 2019, patients of atypical pneumonia due to an unidentified microbial agent were reported in Wuhan, Hubei Province, China. Subsequently, a novel coronavirus was identified as the causative pathogen which was named SARS-CoV-2. As of 12 February 2020, more than 44 000 cases of SARS-CoV-2 infection have been confirmed in China and continue to expand. Provinces, municipalities and autonomous regions of China have launched first-level response to major public health emergencies one after another from 23 January 2020, which means restricting movement of people among provinces, municipalities and autonomous regions. The aim of this study was to explore the correlation between the migration scale index and the number of confirmed coronavirus disease 2019 (COVID-19) cases and to depict the effect of restricting population movement. In this study, Excel 2010 was used to demonstrate the temporal distribution at the day level and SPSS 23.0 was used to analyse the correlation between the migration scale index and the number of confirmed COVID-19 cases. We found that since 23 January 2020, Wuhan migration scale index has dropped significantly and since 26 January 2020, Hubei province migration scale index has dropped significantly. New confirmed COVID-19 cases per day in China except for Wuhan gradually increased since 24 January 2020, and showed a downward trend from 6 February 2020. New confirmed COVID-19 cases per day in China except for Hubei province gradually increased since 24 January 2020, and maintained at a high level from 24 January 2020 to 4 February 2020, then showed a downward trend. Wuhan migration scale index from 9 January to 22 January, 10 January to 23 January and 11 January to 24 January was correlated with the number of new confirmed COVID-19 cases per day in China except for Wuhan from 22 January to 4 February. Hubei province migration scale index from 10 January to 23 January and 11 January to 24 January was correlated with the number of new confirmed COVID-19 cases per day in China except for Hubei province from 22 January to 4 February. Our findings suggested that people who left Wuhan from 9 January to 22 January, and those who left Hubei province from 10 January to 24 January, led to the outbreak in the rest of China. The ‘Wuhan lockdown’ and the launching of the first-level response to this major public health emergency may have had a good effect on controlling the COVID-19 epidemic. Although new COVID-19 cases continued to be confirmed in China outside Wuhan and Hubei provinces, in our opinion, these are second-generation cases. On 31 December 2019, Wuhan, China reported an outbreak of atypical pneumonia caused by SARS-CoV-2 and cases have been exported to other Chinese provinces, as well as internationally [The virus has spread to all prefectures of Hubei province outside Wuhan and all provinces of China have also reported cases outside of Hubei province. Currently, most cases are related to Wuhan and others were caused by patients from Wuhan .Following the confirmed COVID-19 cases reported in Zhejiang province, Guangdong province, Shanghai and other provinces in China reported COVID-19 cases one after another from 20 January 2020.To control the epidemic of COVID-19, Wuhan Municipal People's Government issued a notice saying that citizens should not leave Wuhan for no special reason and the airport and train station from Wuhan corridor were temporarily closed from 23 January 2020 which called ‘Wuhan lockdown’. In addition, Wuhan's urban bus, subway, ferry and long-distance passenger transportation were suspended . ZhejianThe behaviour of the travellers leaving the city of residence for a short time and moving into other cities is migration . Big datWe conducted this study to explore the correlation between migration scale index and the number of confirmed COVID-19 cases. At the same time, we depict the effect of ‘Wuhan lockdown’ and Hubei province launching first-level response to major public health emergencies on the control of COVID-19 epidemic.http://qianxi.baidu.com/). The migration scale indexes of Wuhan and Hubei provinces from 1 January 2020 to 12 February 2020 were used in this study.Data of confirmed COVID-19 cases were extracted from the official websites of the National Health Commission of the People's Republic of China and Health Committee of provinces. Data of migration scale index were extracted from Baidu migration  established by the National Health Commission of the People's Republic of China .P < 0.05. Based on the incubation period of illness from MERS and SARS coronaviruses, CDC believes that symptoms of COVID-19 infection occur within 2–14 days following infection [SPSS 23.0 software was used for Pearson correlation analysis and the significance level used was nfection . The dianfection  and due P < 0.001) and since 26 January 2020, Hubei province migration scale index has dropped significantly . New confirmed COVID-19 cases per day in China except for Wuhan gradually increased since 24 January 2020, and showed a downward trend from 6 February 2020. New confirmed COVID-19 cases per day in Wuhan continued to increase since 27 January 2020, and maintained at a high level without a downward trend. New confirmed COVID-19 cases per day in China except for Hubei province gradually increased since 24 January 2020, and maintained at a high level from 24 January 2020 to 4 February 2020, then showed a downward trend. New confirmed COVID-19 cases per day in Hubei province continued to increase since 24 January 2020, and maintained at a high level without a downward trend , occurred in Wuhan, Hubei province, China –16. The The SARS-CoV-2 has impacted multiple countries, caused severe illness and sustained person-to-person transmission making it a concerning and serious public health threat . Previou24 January 2020 is the Chinese New Year. It is a tradition for Chinese people that migrant workers return home before the Spring Festival. According to the mayor of Wuhan, more than 5 million people have left the city because of the Spring Festival and the outbreak of COVID-19 [To control the epidemic of COVID-19, Wuhan Municipal People's Government issued a notice saying that citizens should not leave Wuhan for no special reason and the airport and train station from Wuhan corridor were temporarily closed from 23 January 2020 which was called ‘Wuhan lockdown’. In addition, Wuhan's urban bus, subway, ferry and long-distance passenger transportation were suspended . SubsequOur study also found that Wuhan migration scale index from 9 January to 22 January, 10 January to 23 January and 11 January to 24 January was correlated with the number of new confirmed COVID-19 cases per day in China except for Wuhan from 20 January to 2 February. This suggests that people who left Wuhan from 9 January to 24 January may led to the outbreak in China except for Wuhan. Meanwhile, our study found that Hubei province migration scale index from 10 January to 23 January and 11 January to 24 January was correlated with the number of new confirmed COVID-19 cases per day in China except for Hubei province from 20 January to 2 February. This suggests that people who left Hubei province from 10 January to 24 January may lead to the outbreak in China except for Hubei province.Calculated from 24 January 2020, after a long incubation period it is 7 February 2020. Our study found that new confirmed COVID-19 cases per day in China except for Wuhan showed a downward trend from 6 February 2020 and new confirmed COVID-19 cases per day in China except for Hubei province showed a downward trend from 4 February 2020. This may indicate that ‘Wuhan lockdown’ and Hubei province launching first-level response to major public health emergencies have had a good effect on the control COVID-19 epidemic. This may also indicate the occurrence of second-generation cases because there were still new confirmed COVID-19 cases in China except for Wuhan and Hubei provinces after 7 February 2020.In spite of the above findings, the limitations in our study should be considered. First, the migration scale of Wuhan and Hubei provinces should include migration to other countries. However, our study only included the number of new COVID-19 cases in China  and didn't include the number of new COVID-19 cases abroad. Second, the number of onset per day should be used in the natural process of disease research. However, the number of new confirmed COVID-19 cases per day was used in this study. Third, we did not explore the correlation between the number of moving into each province from Wuhan or Hubei and the number of new confirmed COVID-19 cases in each province, we will conduct further work to explore the correlation between the number of moving into each province from Wuhan or Hubei and the number of new confirmed COVID-19 cases in each province.Our study found that people who left Wuhan from 9 January to 22 January, and those who left Hubei province from 10 January to 24 January, led to the outbreak in the rest of China. The ‘Wuhan lockdown’ and the launching of the first-level response to this major public health emergency may have had a good effect on controlling the COVID-19 epidemic. Although new COVID-19 cases continued to be confirmed in China outside Wuhan and Hubei provinces, in our opinion, these are second-generation cases.