Rabies is a globally prevalent viral zoonosis that causes 59,000 deaths per year and has important economic consequences. Most virus spread is associated with the migration of its primary hosts. Anthropogenic dissemination, mainly via the transportation of rabid dogs, shaped virus ecology a few hundred years ago and is responsible for several current outbreaks. A systematic analysis of aberrant long-distance events in the steppe and Arctic-like groups of rabies virus was performed using statistical (Bayesian) phylogeography and plots of genetic vs. geographic distances. The two approaches produced similar results but had some significant differences and complemented each other. No phylogeographic analysis could be performed for the Arctic group because polar foxes transfer the virus across the whole circumpolar region at high velocity, and there was no correlation between genetic and geographic distances in this virus group. In the Arctic-like group and the steppe subgroup of the cosmopolitan group, a significant number of known sequences (15–20%) was associated with rapid long-distance transfers, which mainly occurred within Eurasia. Some of these events have been described previously, while others have not been documented. Most of the recent long-distance transfers apparently did not result in establishing the introduced virus, but a few had important implications for the phylogeographic history of rabies. Thus, human-mediated long-distance transmission of the rabies virus remains a significant threat that needs to be addressed. Lyssavirus. A number of lyssaviruses can cause a lethal disease called rabies [Rabies virus (RABV) is a negative-sense ssRNA virus belonging to the genus d rabies . Howeverd rabies .RABV consists of bat-associated and carnivore-associated phylogenetic groups. Bat-associated RABV circulates only among the New World bats. Carnivore-associated RABV may be found in every continent except Australia and the Antarctic  and consIt has been hypothesized that intercontinental transportation of rabid animals by humans provided the global distribution of cosmopolitan RABV, likely over the last few hundreds of years ,7. HowevExpansion of natural RABV reservoirs is relatively slow. The mean annual virus spread rate depends on the species of the reservoir animal and the ecological region . The ratUnderstanding natural virus dissemination patterns is critical for planning preventive measures. Vaccination of humans and domestic animals is an effective measure against the disease. Furthermore, wildlife vaccination can be used to eradicate virus reservoirs in a large territory. Indeed, some parts of the world, e.g., western Europe, have been declared rabies-free areas . SimultaHistorically, finding an RNA virus with a sequence nearly identical to those of viruses from a distant location unequivocally indicates that a long-distance transfer has occurred. In the case of the rabies virus, the simplest explanation is human involvement in pathogen dissemination. This approach may be called the gold standard in the epidemiological investigation of isolated imported cases. However, it is more challenging to analyze the role of anthropogenic dissemination in pathogen evolution and spreading history as a whole, especially as the number of known sequences grows exponentially and goes beyond the capacity of classical analysis tools.There are several possible approaches to assess the virus spread rate. The first one is based on the dependence between evolutionary distances and the geographical spacing of virus sequences. In that case, all studied objects are analyzed in all possible pairs. Furthermore, evolutionary distances (the proportion of nucleotide sites at which the two compared sequences are different) and geographic distances are calculated for every pair . The resAnother approach, the Bayesian phylogeographic framework, infers virus dissemination velocity within different phylogenetic tree branches. A significant deviation of the inferred spread rates at tree branches would suggest the role of an anthropogenic factor or another possible explanation. This method has two variations: discrete phylogeography that uses isolation locations expressed as fixed categories and continuous phylogeography that uses actual sampling coordinates . A variaIn the current study, we have implemented both the genetic distance-based approach and continuous phylogeography to several RABV groups, i.e., cosmopolitan, Arctic and Arctic-like, to study the role of anthropogenic virus spread on a global scale.Rabies lyssavirus (Taxonomy ID: 11292) were extracted . To provide the best balance of sequence length and number of sequences in the data set, 7616 entries aligning with genome positions 170–1150 in the reference sequence NC_001542 were selected (see below)  . The datData sets for RABV genome coverage visualization were generated using open-source tools . Then, ahttps://raw.githubusercontent.com/AndreiDeviatkin/gene_geo/main/data/fasta/txid11292_260620_950%2B_210-1140_cut_al_final.fas, assessed on 28 December 2021).Maximum likelihood (ML) phylogenetic inference  was perfn = 2423) and Arctic-related  RABV sequences were extracted from the whole data set. Furthermore, vaccine-associated sequences were omitted. Arctic and Arctic-like groups were analyzed separately due to suggestively different ecological traits. The steppe subgroup of cosmopolitan viruses was also selected for a separate analysis as one of the best-explored subgroups with a known anthropogenic transfer event.Then, cosmopolitan (n = 1824), steppe subgroup of cosmopolitan (n = 205), Arctic (n = 453) and Arctic-like (n = 201). Identical sequences with an identical place of sample collection were omitted in the Arctic group before Bayesian analysis, so only 292 out of 453 sequences were used.Finally, only sequences with known dates and locations of sample collection were selected, resulting in the following data sets: cosmopolitan  approach implemented in BEAST 1.10.4 . The BEAMaximum clade credibility (MCC) trees were inferred under the relaxed random walk model with a Cauchy distributed diffusion rate variation among branches, which suggests heterogeneity in branch velocities. We used a GTR substitution model with the uncorrelated relaxed log-normal molecular clock assumption  and BayeA map with the locations of sample collection D was genhttps://rpubs.com/andreideviatkin/RABVdistribution (assessed on 28 December 2021). In order to avoid the overlap of isolates from the same region, a slight random noise was introduced into the coordinates used in this map.An interactive map with labeled markers is available at https://github.com/AndreiDeviatkin/gene_geo (assessed on 28 December 2021). This code was implemented in the R shiny webserver as an online tool available at https://andreideviatkin.shinyapps.io/gene-geo/ (assessed on 28 December 2021).Genetic and geographic distance concordances (Gene-geo plots) were visualized in the R environment. Gene-geo plots have not demonstrated consistent statistical patterns for tested data sets. The source code is available at n = 292 and Arctic-like, n = 201), the globally prevalent and well-explored cosmopolitan group (n = 1824), and its steppe subgroup (n = 205), which are well-represented in GenBank and have well-characterized virus trafficking rates [The genome coverage plot indicated that RABV genome fragments were not evenly represented in GenBank . A 981 nng rates  and a knng rates .Calculation of a Bayesian phylogeographical framework grows with the number of sequences and is not practical for such large data sets. Furthermore, the accuracy of Bayesian phylogeographic models for large data sets remains unclear, as these models tend to perform best at intermediate sequence data set sizes . Thus, tA gene-geo plot of cosmopolitan RABV demonstrated a fair correspondence between genetic and geographic distances. This relationship was not linear, likely due to mutation saturation at about 10% nucleotide sequence distance and the impossibility of gradual virus spread between continents. The plot contained several sequence pairs that obviously deviated from the general distribution of pairwise values (The gene-geo plot for Arctic RABV demonstrated a limited concordance between genetic and geographic distances distribution patterns C. This lThe distance-based method (gene-geo plot) was tested in parallel with the Bayesian phylogeographic approach for the other data sets, i.e., the steppe subgroup   and the n = 205) was 4.13 × 10−4 (95% HPD 3.24 × 10−4–5.00 × 10−4) nucleotide substitutions/site/year (s/s/y). This is concordant with the estimates of RABV N-gene substitution rates made for other subgroups of carnivore RABV: 3.89 × 10−4 s/s/y (2.88 × 10−4–4.96 × 10−4) [−4 (2.10 × 10−4–2.80 × 10−4) s/s/y [−4 (2.62 × 10−4–5.02 × 10−4) s/s/y [−4 (1.82 × 10−4–3.12 × 10−4) s/s/y [−4 (3.94 × 10−4–6.68 × 10−4) s/s/y [The mean nucleotide substitution rate inferred using the N-gene data set for steppe subgroup ( × 10−4) , 2.44 × 4) s/s/y , 2.47 × 4) s/s/y , 5.23 × 4) s/s/y . This coThe gene-geo plots for both the Arctic-like and steppe groups were compatible with recent long-distance virus transfers evident as dots with low genetic and high geographic distances A. The phThe most vivid examples of sequences classified as “cosmopolitan” or “arctic-like” that had evidence of long-distance transfers upon gene-geo analysis are summarized in In general, RABV is considered a typical object for phylogeographic studies . HoweverWhile the Bayesian frameworks converged for other RABV groups, potential human-mediated transfers were a ubiquitous factor to be considered according to the gene-geo plots. We used a stringent cut-off of 500 km/year and a comparable genetic distance criterion to define likely anthropogenic transfers. This approach detected several known events and a comparable number of undescribed ones . OverallIt has been discussed previously that the history of RABV was significantly shaped by human activity. The worldwide dissemination of the carnivore-associated group of RABV was associated with colonial expansion and international trade accompanied by uncontrolled dog transportation that occurred in the 15th–19th centuries . The rolIn the cosmopolitan group, all virus pairs spaced by over 8000 km also differed by at least 5% in the nucleotide sequence. Extrapolating RABV substitution rates identified here and elsewhere (see above), this corresponds to 100–250 years of circulation after the most recent common ancestor. Thus, virus control measures in the last century were generally efficient to prevent virus spread between the Old and New World, and intercontinental RABV transfer is not a major factor in virus evolution nowadays.The majority of recent long-distance virus trafficking found in this study occurred within Eurasia . MoreoveThe two methods for the detection of aberrant long-distance virus transfers produced comparable results but had significant differences. Statistical phylogeography found many known and unreported long-distance transfer events but failed in the cases with an absent phylogeographic signal (Arctic RABV) or a too high number of sequences (cosmopolitan group). Gene-geo plots were applicable in both these cases to detect either a few long-distance transfers in the cosmopolitan group A or to cA major limitation of the large-scale phylogeographic analysis is the lack of precise virus sampling coordinates in GenBank records. Dedicated studies can use exact sampling location data; however, most published sequences have just country or, at best, province data. Many regions are hundreds of kilometers across, and virus isolation coordinates had to be approximated. Transfers between such regions could appear more rapid than they actually were. On a global scale, we observed a fair correlation between genetic and geographic distances. However, a more precise analysis, such as inferring virus trafficking rates, may not be reliable with the available data. In addition, for this reason, we abstained from analyzing less obvious potential long-distance transfers. Clearly, precise sampling coordinates would significantly increase the value of sequence data.Another general limitation to ecological studies using RABV genomic data comes from the over-representation of cases of medical or veterinary importance in sequence databases. These events generally represent endemic viruses in the area but are, in most cases, dead-end events in virus evolution. Indeed, most long-distance virus transfers described here did not result in the establishment of novel virus lineages in distant regions. Only one suggestive transfer that occurred less than 70 years ago actually significantly impacted steppe RABV phylogeography (indicated by a star in The human-mediated spread of rabies is a significant threat (reviewed in ) and req


In the battle to quickly identify potential yellow fever arbovirus outbreaks in the Democratic Republic of the Congo, active syndromic surveillance of acute febrile jaundice patients across the country is a powerful tool. However, patients who test negative for yellow fever virus infection are too often left without a diagnosis. By retroactively screening samples for other potential viral infections, we can both try to find sources of patient disease and gain information on how commonly they may occur and co-occur. Several human arboviruses have previously been identified, but there remain many other viral families that could be responsible for acute febrile jaundice. Here, we assessed the prevalence of human herpes viruses (HHVs) in these acute febrile jaundice disease samples. Total viral DNA was extracted from serum of 451 patients with acute febrile jaundice. We used real-time quantitative PCR to test all specimens for cytomegalovirus (CMV), herpes simplex virus (HSV), human herpes virus type 6 (HHV-6) and varicella-zoster virus (VZV). We found 21.3% had active HHV replication , and that nearly half (45.8%) of these infections were characterized by co-infection either among HHVs or between HHVs and other viral infection, sometimes associated with acute febrile jaundice previously identified. Our results show that the role of HHV primary infection or reactivation in contributing to acute febrile jaundice disease identified through the yellow fever surveillance program should be routinely considered in diagnosing these patients. Though these investigations associated 224 suspected yellow fever cases with a potential causative pathogen, many remained unidentified. Here, we investigated four human herpes viruses (HHVs) known sometimes to cause acute jaundice and which infect African populations at high prevalence: herpes simplex virus (HSV1 & HSV2), varicella-zoster virus (VZV), cytomegalovirus (CMV), and human herpes virus type 6 (HHV-6) [exanthema subitum  [Due to the severity and the high risk of re-emergence of yellow fever, a disease caused by an arthropod-borne virus, the Democratic Republic of the Congo (DRC) has implemented an active syndromic surveillance program throughout the country since 2003 based on the involvement of healthcare structures located in its health areas. All suspected clinical cases compatible with Yellow fever disease, including fever and jaundice, were notified to the Ministry of Health and rapidly tested for Yellow fever infection. A total of 652 suspected YF cases were detected through the program, of which only less than 5% tested positive for YFV. In order to investigate other causative pathogens, we previously investigated other viral pathogens, sometimes associated with acute febrile jaundice, retrospectively in available samples ,2. These.4% n = 3/365 test2.3% n = /379 and 2.3% n = 5/470, 2.nfantum) ,12. Yellow fever surveillance is an operational surveillance program based on a case definition of the infection. Suspected cases of yellow fever are defined as patients with an acute onset of fever followed by jaundice within two weeks of the first symptoms, and that are negative for malaria by thick blood smear or fail to respond to appropriate anti-malarial treatment. Other clinical signs are generally not recorded. The data collection form only includes demographic data such as age, gender, date of collection and health zone of origin ,2.® Advanced XL Biorobot  as described previously [Total viral DNA was extracted from 200 µL of serum and eluted in 120 µL of elution buffer using the EZ1We used real-time quantitative PCR with previously published primers and probes to test all specimens for CMV, HSV, HHV-6 and VZV ,14,15. Ap < 0.05. Differences in HHV infection prevalence (at least one HHV detected among those sampled) between sexes and age  were tested using chi-square tests (using stats function ‘chi.test’); p-values calculated on 1 and 2 degrees of freedom, respectively. Co-infections were evaluated where possible from the 451 samples for which the presence of all target HHVs were tested, and represent only a minimum number of co-infections with other target hepatitis virus and arbovirus infections, as complete data for these were not available for all 451 samples. The influence of sex, age (in years), and their interaction on relative risk of co-infection (mono-HHV infection vs. HHV + co-infection) was tested using a logistic regression  followed by analysis of deviance  using the 287 samples from which data were available for all target viruses. The interaction term was removed from the model when the initial test revealed that any interaction was not statistically significant .All statistical analyses were performed in the R statistical software environment (version 4.0.5), and statistical significance was evaluated at We detected DNA for 109 human herpes virus infections in 96 of the 451 samples collected from January 2003 to January 2012 . χ2 < 0.01, df = 1, p = 0.99) and age groups   or arboviruses , and 5 (5.2%) infected by both multiple herpes viruses and at least one additional target virus  nor age .Among these herpes virus positive samples, 59 (13.1%), 11 (2.4%), 28 (6.2%) and 11 (2.4%) were positive for CMV, HSV, HHV-6 and VZV, respectively. HHVs were detected in samples without significant differences between sex ( = 0.33) A. Intere = 0.33) B. Furtheet virus B. The riThis study shows that many HHVs were detectable in the blood of acute febrile jaundice patients, as 21.3% (96/451) had a detectable virus for at least one HHV tested. This frequency is most certainly underestimated because detection of viral DNA in blood does not necessarily reflect the amount of virus produced by active infection of distant lymphoid tissue and organs , and viral DNA could be highly fragmented making it difficult to detect by PCR  ,17. NearThe origin of HHV viral DNA in serum can be a primary infection but is more likely a reactivation of latent virus induced by co-infections highlighted in our study, particularly for viruses such as CMV which are acquired at high seroprevalence rates in childhood. The specific mechanisms for reactivation of HHV are not well known and few data exist to our knowledge on the clinical impacts of co-infection with HHV and hepatitis viruses or arboviruses . The majDiagnosis of acute febrile jaundice remains a challenge in yellow fever surveillance. Our study indicates active herpes virus infection in 21.3% of acute febrile jaundice patients, with nearly half of the infections characterized by the co-detection of multiple active viruses, each known sometimes to produce acute febrile jaundice symptoms These multiple infections may partly explain symptoms compatible with yellow fever disease. These findings should therefore be systematically taken into account in the therapeutic management and the delivery of appropriate palliative care to patients exhibiting yellow fever-like disease. Future studies should address the prevalence of HHVs and co-infections in the general population, in order to fully understand the implication of our results.


The Mn2+ concentration in PJ produced in different years was defined using Atomic Absorption Spectrometry. The optimal Mn2+ concentration and the amount of PJ, were estimated in an in-vitro analysis, and were then prospectively tested in a population of patients who underwent MRCP. The results were compared with those achieved with the previous standard amount of PJ used in a similar population. The concentrations of Mn2+ in commercial PJ produced in different years did not differ. A total amount of 150 ml (one glass) of PJ having a high Mn2+ content (2.37 mg/dl) was sufficient for the suppression of the GD liquid signal, despite the additional dilution caused by GD liquids since it led to a final concentration of Mn2+ of 0.5–1.00 mg/dl. The optimized single-dose oral administration of 150 ml (approximately one glass) of PJ having a high Mn2+ concentration prior to MRCP was adequate to guarantee the correct amount of Mn2+ to suppress the GD signal.To evaluate the potential variability of Manganese (Mn Currently, MRCP has innumerable indications, such as evaluation of the normal biliary anatomy and its variants, and congenital and acquired biliary pathologies2.Magnetic Resonance Cholangiopancreatography (MRCP) is the first-line non-invasive imaging technique for evaluating the biliary tract if immediate therapy for a known problem is not the primary aim3.Magnetic Resonance Cholangiopancreatography is performed with heavily T2-weighted (T2W) sequences, exploiting the fact that bile, as do all static fluids, has a long T2 relaxation time, making the bile ducts hyperintense and well recognisable (white) from the decreased signal of the background tissues (black), such as solid organs and flowing blood5. Several oral negative contrast agents, containing paramagnetic substances which shorten the T2 relaxation time, thus reducing the signal hyperintensity of gastro-enteric fluids in standard MRCP sequences, have been produced by pharmaceutical companies and have been utilised in daily radiological practice in the past in order to overcome this limitation7. However, these negative oral contrast agents have many limitations; they are relatively unpalatable, are too diluted in the gastrointestinal tract or are too expensive6. To overcome these limitations, in more recent years, natural fruit juices have gained attention, the most popular being pineapple juice (PJ) and blueberry juice, owing to their properties which are similar to pharmaceutical oral negative contrast agents, having the same ability to suppress the high signal from gastrointestinal tract liquids on MRCP, but without the described limitations, having good palatability and a lower cost.A recognised limitation of MRCP is the possible overlap of the static fluids in the pancreatobiliary system and the static fluid in the gastrointestinal tract  which can obscure the distal portion of the common bile duct or simulate a disease2+) concentration and not on their Iron (III) concentration8. Moreover, it has also been demonstrated that all the commercial juice had a Mn2+ concentration capable of suppressing the gastrointestinal liquid signal on MRCP, and that PJ represents the optimal natural substance for suppressing gastrointestinal hyperintensity in heavily T2W sequences of MRCP due to its high Mn2+ content8.The ability of commercially available fruit juice to shorten the T2 relaxation time has recently been demonstrated to be closely dependent only on the Manganese  liquid signal.This study has attempted to answer these three open issues. In particular, the primary aim of this study was to evaluate the potential variability of the Mn2+ in PJ of the same commercially available brand produced in different years, redefined using AAS, did not demonstrate any difference. The absolute Mn2+ concentration measured was 2.37 mg/dl. This value did not differ with that measured in a previous study; in fact, the Mn2+ concentration in the PJ previously determined was 2.38 mg/dl8.The concentrations of Mn2+ concentrations (A–E) (from 1.00 to 0.063 mg/dl) is reported in Table 2+ concentration ranging from 0.5 to 1.0 mg/dl  the best suppression of the GD liquid signal was obtained with a Mn2+ concentration ranging from 0.5 to 1.0 mg/dl. Assuming that the total GD fasting content does not exceed 200 ml, 150 ml of PJ (1 full glass) containing 2.37 mg/dl of Mn2+, when diluted in GD liquids, will reach an overall Mn2+ concentration of approximately 1 mg/dl.For the clinical validation, the Authors relied on the results of the in vitro measurements: (a) 150 ml (1 full glass) of PJ contain 2.37 mg/dl of MnP = 0.936) and age .The new selected dose of 1 glass of PJ was orally administered to n. 308 fasting patients (Group 1) who consecutively underwent MRCP. The results were compared to 305 patients (49.8% of the entire study population) which underwent MRCP after the oral intake of 300 ml of PJ (Group 2). The two groups were not different regarding gender .There were no statistical differences in the degree of suppression of the GD liquid signal between the two groups of patients after the analysis of the MRCP images Fig. ; Table 3P = 0.455). No difference was observed regarding SI of GD fluid content in the stomach (SIS) and in the duodenum (SID) after oral administration of PJ between the two groups of patients  of the reference liquid  (SIR) did not showed any difference between the two group of patients .The overall quality of the MRCP images did not differ between the two groups as reported in Table 10. A recognised limitation of MRCP is represented by the possible overlap of the static liquids in the pancreatobiliary system and those in the gastrointestinal tract  which can obscure distal biliopancreatic ducts or simulate a disease having the same signal intensity on MRCP5.In recent years, considerable progress has been made in improving the quality of MRCP, allowing evaluation of both the normal biliary anatomy and its variants, and congenital and acquired benign and malignant biliary diseases using this imaging technique2+ alone represents the key element in fruit juices for suppressing the GD liquid signal and not Iron (III) as had previously been hypothesised8. Despite these advances, some open questions remain, In fact, it is unknown whether there is a variability in terms of the Mn2+ concentration in commercially available PJ of the same brand produced in different years. Furthermore, to date, the exact minimum concentration of Mn2+ and the correct amount of juice sufficient for obtaining signal suppression of the GD liquids after additional dilution in the stomach and in the duodenum in fasting patients prior to MRCP has not been analysed or standardised.Strong efforts have also been made to improve MRCP image quality and patient acceptance, such as the use of fruit juice as an oral negative contrast agent in order to overcome the limitations of oral chemical contrast media represented by their poor palatability and high cost. A recent paper has shown that Mn2+ concentration in PJ of the same commercially available brand produced in different years. In the previous study, it had been demonstrated that there was no variability between PJ of different batches but of the same brand8. The combination of these two results is very important for standardisation regarding the adoption of PJ anywhere in the world since the concentration of Mn2+ will always be the same in the different batches of the same brand, regardless of the year of production of the PJ.First of all, the present data showed the absence of variability in Mn2+ which permitted obtaining the best results in suppressing the GD liquid signal ranged from 0.5 to 1 mg/dl.Secondly, not previously analysed in the scientific literature, this study started with an in-vitro analysis showing that the concentration of Mn11, the amount which should be added to a similar quantity of the fasting duodenal content. Therefore, the optimal amount of PJ to be administered orally before MRCP is that which allows obtaining a Mn2+ concentration of 0.5–1 mg/dl after additional dilution of the PJ caused by a mean total amount of 200 ml of GD liquids. The present study revealed that the oral administration, half an hour prior to MRCP, of only 1 glass (150 ml) of commercially available PJ, provided that it had a high Mn2+ content (2.37 mg/dl) was sufficient to obtain excellent suppression of the GD liquid signal without affecting the image quality of the examination. According to the best of the Authors’ knowledge, there are no previous studies which have analysed this topic. This amount was very important for the following reasons: (a) the amount of PJ administered orally is half of the dose previously recommended (2 glasses)8, thus halving the cost (b) the patient has to drink only one glass of juice which this can be of less discomfort in patients who find it difficult to drink for clinical reasons or because they do not like to drink pineapple juice due to its taste and (c) in the pandemic era, a single-patient/single-dose within a sealed Tetra Pak package is recommended in order to avoid contamination during the pouring of juice from a 1-l bottle of juice into a glass.Lastly, the Authors considered the obvious dilution of ingested PJ in the GD content after fasting; the data related to the volume of liquids within the gastric lumen in a healthy subject after a 10-h fasting period were investigated, having a reported maximum volume of 96 mlThe clinical data of the present study were also important because they were tested on a large study population, including patients prospectively and consecutively enrolled in order to evaluate this new dosage for all possible clinical indications for MRCP. When comparing the two study groups, there were no statistically significant differences in terms of indications for the examination in the patients who received 1 glass of PJ prior to MRCP and those who received 2 glasses. Moreover, just as importantly, in more than 97% of cases, the assumption of PJ did not affect the overall quality of the MRCP. Finally, the oral assumption of only 1 glass of PJ can be recommended before performing MRCP for any indication without the risk of incurring a reduction in the image quality.The main limitation of the present study was the non-prospective comparison between patients who assumed 1 glass of PJ (prospectively enrolled) and those who assumed 2 glasses of PJ (retrospectively collected). However, the necessary statistical tests were carried out to correctly identify any possible differences between the two groups without identifying any differences between the two populations in terms of demographic characteristics and indications for MRCP.2+ content of PJ of the same commercially available brand produced in different years. The Mn2+ concentration necessary to suppress the intensity of the GD liquid signal on MRCP ranged from 0.5 to 1.0 mg/dl. The oral administration of 150 ml (one glass) of PJ containing a high concentration of Mn2+ prior to MRCP is sufficient to guarantee the correct Mn2+ concentration to suppress the GD liquid signal, regardless of its additional dilution by gastrointestinal liquids.There was no variability in the MnThis study describes a standard of care change and was approved by the Institutional Review Board (Comitato Etico di Area Vasta Emilia Centro della Regione Emilia-Romagna) with registration number 791/2020/Oss/AOUBo (date of first registration: 11/05/2020). Written informed consent was obtained from all patients. This study was conducted according to the Declaration of Helsinki for clinical studies.2+ content in PJ of the same commercially available brand produced in different years and to identify the optimal concentration of Mn2+ in the correct amount of PJ to be orally administered to fasting patients prior to MRCP in order to suppress the GD liquid signal.In vitro measurements were carried out to evaluate the potential variability of the MnSubsequent clinical validation was carried out to assess the secondary aim of the present study.8.All the MRCP examinations were carried out according to the standardised protocol described in detail in a previously published paper2+ in PJ of the same commercially available brand previously identified as having the highest Mn2+ content (2.38 mg/dl)8 were additionally redefined to exclude the possible variability of different periods of pineapple cultivation produced in different years utilising Atomic Absorption Spectrometry (AAS) with a Thermo iCE3300 AA spectrometer and acetylene/air flame atomisation.The concentrations of Mn2+ solution with different dilutions to identify the Mn2+ concentration sufficient to suppress the signal of intestinal liquids on MRCP. In particular, five solutions were prepared. A dose of 76.9 mg of Mn2+ sulphate monohydrate was weighed and dissolved in 500 ml of distilled water obtaining a solution which contained 5.00 mg/dl of Mn2+. Thereafter, 100 ml of this solution was diluted to 500 ml obtaining solution A containing 1.00 mg/dl of Mn2+. Additional dilutions were prepared to obtain solutions with decreasing concentrations of Mn2+: solution B with 0.50 mg/dl of Mn2+, solution C with 0.250 mg/dl of Mn2+, solution D with 0.125 mg/dl of Mn2+ and solution E with 0.063 mg/dl of Mn2+ 8 was chosen for the in vivo study.The concentration of Mn2+ solutions and the diluted PJ previously selected as having the highest Mn2+ content (2.38 mg/dl)8. Therefore, the PJ selected was diluted into different solutions to obtain the same Mn2+ concentration as the pure Mn2+ solutions. In particular, 85 ml of this PJ was diluted to 200 ml to obtain a Mn2+ concentration of 1 mg/dl (solution F). Additional dilutions permitted obtaining solutions G (0.5 mg/dl Mn2+), H (0.25 mg/dl Mn2+), I (0.125 mg/dl Mn2+) and J (0.063 mg/dl Mn2+). For these five solutions, the values of the T2W sequences were determined  was orally administered to fasting patients who consecutively underwent MRCP for any indication half an hour prior to the examination. The results were then compared with those of a previous population of patients who underwent MRCP between June 2018 and December 2018 in whom a standard dose of PJ of 300 ml (two glasses) was administered orally, as indicated in a previous studyTwo experienced radiologists (MR and SB) with more than ten years of experience in hepatobiliary and pancreatic imaging, blinded to the amount of PJ administered prior to MRCP, evaluated the results in a blinded fashion. A comparison between Group 1 (one glass) and Group 2 (two glasses), was carried out, based on the evaluation of the degree of reduction in the intensity of the GD liquid signal in the standard MRCP sequences. The suppression of the GD liquid signal was graded using a four-point scale: Grade 1 ; Grade 2 ; Grade 3  and Grade 4 .The radiologists also evaluated the quality of all the MRCPs for each patient to avoid any possible impact of the PJ ingestion on the overall quality of the examination. The quality of the images, acquired after PJ ingestion, was graded using a four-point scale: Grade 1 ; Grade 2 ; Grade 3  and Grade 4 .3 was measured in 3 different sites: in the second portion of the duodenal lumen, in the region of the gastric fundus lumen and in correspondence of a reference liquid, meaning the location where the highest liquid intensity could be measured for each patient .In an attempt to perform an image processing quantification of GD liquid signal suppression, the value of SI of a region of interest (ROI) of 0.5 cm2-test or the Fisher’s exact test (2-tailed) for the qualitative variables while the Mann–Whitney test was carried out for the quantitative variables. A P value of less than 0.05 was considered statistically significant. All the analyses were carried out using SPSS 23.0 software .The distribution asymmetry of the quantitative data was assessed using the Skewness test. The quantitative variables were expressed as mean ± standard deviation, or median and interquartile range (IQR), or ratio as appropriate. Comparisons between the groups were carried out using the χInstitutional Review Board (Comitato Etico di Area Vasta Emilia Centro della Regione Emilia-Romagna) approval was obtained with registration number 791/2020/Oss/AOUBo (date of first registration: 11/05/2020). This study was conducted according to the Declaration of Helsinki for clinical studies.Prospective study describing a standard of care change, carried out at a single institute.Written informed consent was obtained from all patients.


Some viruses have the ability to block or suppress growth of other viruses when simultaneously present in the same host. This type of viral interference or viral block has been suggested as a potential interaction between some respiratory viruses including SARS-CoV-2 and other co-circulating respiratory viruses. We explore how one virus' ability to block infection with another within a single host affects spread of the viruses within a susceptible population using a compartmental epidemiological model. We find that population-level effect of viral block is a decrease in the number of people infected with the suppressed virus. This effect is most pronounced when the viruses have similar epidemiological parameters. We use the model to simulate co-circulating epidemics of SARS-CoV-2 and influenza, respiratory syncytial virus (RSV), and rhinovirus, finding that co-circulation of SARS-CoV-2 and RSV causes the most suppression of SARS-CoV-2. Paradoxically, co-circulation of SARS-CoV-2 and influenza or rhinovirus results in almost no change in the SARS-CoV-2 epidemic, but causes a shift in the timing of the influenza and rhinovirus epidemics. A novel coronavirus first detected in Wuhan, China in late 2019 spread rapidly around the world  and exposure to the second (SARS-CoV-2) will temporarily prevent infection with the second virus (SARS-CoV-2), but infection with the second virus (SARS-CoV-2) and exposure to the first (influenza) will lead to coinfection. We find that this type of interaction leads to a smaller epidemic of the suppressed virus, but can also alter the timing of the epidemic of the dominant virus.Spread of an infectious disease through a population is often modeled using ordinary differential equation (ODE) compartment models. The common basis for many of these epidemiological models is the Susceptible-Exposed-Infectious-Recovered (SEIR) model where each class of the population is represented by a compartment and movement of people between compartments is governed by ODEs . People can be infected by either virus with force of infection βi  and move into the exposed compartments (Ei). Our assumption is that virus 1 (non-SARS-CoV-2 virus such as influenza) blocks infection with virus 2 (SARS-CoV-2) within the host, so people exposed to virus 1 transition to the infectious compartment (I1). Upon recovery from virus 1, they are immune to virus 1, but are now susceptible to virus 2 (R) and immune to both viruses. People who are infected first with virus 2 can develop a coinfection. Thus, people in both the exposed and infected compartments for the second virus (E2 and I2) can become coinfected. We assume that coinfected exposed (E3) people have moved to the infected phase for virus 2, so can infect others with this virus, but are still only exposed to virus 1. After some transition time, they become fully coinfected (I3) and capable of transmitting both viruses. After some time, coinfected patients also recover and are immune to both viruses. Those patients exposed to virus 2 who do not become coinfected move to the infected compartment (I2) and recover. At this point, they are immune to virus 2, but are still susceptible to virus 1 . We do, however, explore the possibility that coinfection can lead to a longer recovery time.We make a number of simplifying assumptions to keep the model tractable. We assume that the population remains fixed, with total population size 3. Since the recovery rates for SARS-CoV-2 coinfections are unknown, we consider both a faster and slower coinfection recovery rate: δ3= 0.1 /d or δ3= 0.01 /d. Simulations are run using python's odeint differential equation solver  to assess which model parameters contribute most to changes in epidemic outcome. We allow parameter values to range between ±10% of their base value and use 1,000 different randomly selected parameter combinations to calculate the PRCC. The partial rank correlation coefficient is close to ±1 if there is a high degree of correlation between the independent and dependent variables, with positive values indicating a positive correlation (both increase or decrease together) and negative values indicating a negative correlation. The output variables examined for PRCC are total number of virus 1 infections, total number of virus 2 infections, and the number of coinfections.Ii(0) = 100. We assume an initial susceptible population, S(0), of 331,002,651 (the population of the US). All other classes are initially set to zero. We start by examining the interaction of two identical viruses using parameters for influenza infection . In the model, people infected with virus 1 cannot be infected by virus 2, whereas people infected with virus 2 can be coinfected. The model also allows for sequential infections without any asymmetry, i.e., a person can get the other virus after recovering from their initial infection. Infection is initiated by 100 infected individuals for each of the viruses, nfection .1 and δ1), and the total number of people infected with virus 2 depending most strongly on virus 2 infection and recovery rates (β2 and δ2). The asymmetry in the model is most apparent when looking at the parameter dependence of the total number of coinfections. The total number of coinfections depends more strongly on the infection rate and recovery rate of the second virus than on the infection and recovery rates of the first virus. The only way to become coinfected is to first be infected with the second virus, so the more virus 2 infections and the longer they last, the more likely we are to see coinfections.We use partial rank correlation coefficient (PRCC) to assess how different model parameters affect the outcome of the epidemic. We consider three outcomes: the total number of people infected with virus 1, the total number of people infected with virus 2, and the total number of coinfections. Results of the PRCC are shown in 1 or β2) of either virus changes. We use three measures of the epidemic curve to assess how the epidemic changes: the peak number of infected, the time of peak, and the cumulative number of infected.In reality the viruses that circulate at the same time are described by different epidemiological parameters. To better understand how differences in viral spread enhance or suppress the population-level interaction of the two viruses, we examine the epidemic when the infection rate (β1) or the infection rate of virus 2 (β2). Thus, varying β1 increases the peak number of infected for virus 1  that is not apparent in the response of virus 1 to changes in β2 of the left column. To highlight this asymmetry, we subtract the direct responses (virus i response to change in βi) of virus 1 and virus 2, as well as subtract the indirect responses (virus i response to change in βj) of virus 1 and virus 2, with the results given in 1 or β2). Thus, the within host suppression of one virus by another is reflected in decreased population-level spread of the suppressed virus.There is also very little obvious asymmetry in the graphs; changing the infection rate of the dominant and suppressed virus appears to have more or less the same outcome. However, if we look carefully, there is a small dip in virus 2 response to changes in βBased on the results of a recent modeling study that suggests that SARS-CoV-2 is suppressed by several common respiratory viruses . According to the analysis of section 3.2, the largest changes occur when the two viruses have similar growth rates, so this is why our model predicts the largest effect for the RSV/SARS-CoV-2 combination. In reality, all of these viruses, and more, are co-circulating together, making it difficult to isolate the interaction of one pair (de Celles et al., Two extensions of our interaction model, inclusion of patient death and incomplete viral block, are considered in the Despite these limitations, our model provides insight into how co-circulating viruses will spread through the population if they participate in viral interference at the within-host level. The model suggests a reduction in the total number of cases of the suppressed virus, although the effect is small, but also suggests a shift in the timing of the peak of the dominant virus is possible.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.LP and HD: conceptualization, methodology, validation, and writing—review and editing. LP: software and formal analysis. HD: writing—original draft preparation, supervision, and project administration. Both authors have read and agreed to the published version of the manuscript.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.


The main objectives of this study were to model various scenarios of African swine fever (ASF) virus transmission among farms in Vietnam and to evaluate the impact of control strategies using North American Animal Disease Spread Model (NAADSM). A total of 7,882 pig farms in the Red River Delta (RRD) region were obtained from the General Statistics Office, and then, random points corresponding to the number of farms in each province were generated as exact farm locations were not available. A total of 10 models were developed, including movement control scenarios. In addition, we conducted sensitivity analysis to assess the impact of indirect contact transmission probability (TP). Overall, the indirect contact exhibited an important role in transmitting the ASF virus. In order to minimize ASF transmission between farms, we found that movement restriction needed to reach a certain level (approximately between 50% and 75%) and that the restriction had to be applied in a timely manner. This study offers valuable insight into how ASF virus can be transmitted via direct and indirect contact and controlled among farms under the various simulation scenarios. Our results suggest that the enforcement of movement restriction was an effective control measure as soon as the outbreaks were reported. In addition, this study provided evidence that high standards of biosecurity can contribute to the reduction of disease spread. Since there is no vaccine for ASF, we evaluated the effectiveness of movement control on contact rates by 25%, 50%, 75% and 100% reduction, which was applied to both direct and indirect contact rates. It was hypothesized that movement restrictions were imposed for the baseline scenario within 4 weeks of detection of outbreaks. We conducted sensitivity analysis to assess the impact of infectious duration for small farms (basic: 52 weeks) and indirect contact TP for the small/medium farms (basic: 0.6) by −25%, −50% and −75%, respectively. In addition, the timing of movement restriction was imposed at 2 weeks, 6 weeks and 8 weeks after detection, respectively, under the 25% and 50% movement restriction scenarios.3The baseline scenarios (A1: including both direct and indirect contacts) showed that a total of 7,640  median farms were infected, while A2 (only including indirect contact) presented a slightly lower median number of infected farms  Table . The epiThe sensitivity analysis was implemented to evaluate on infectious duration , TPs for indirect contact , movement restrictions and the timing of restrictions compared to the baseline scenario. We found that the infectious duration did not have an impact on the results . The number of medium infected farms for scenario IC3 had a sharp reduction by 99.99% compared to the baseline scenario (A1) , is used in the baseline scenario and other movement control scenarios, which was based on our assumption owing to the above considerations. Our indirect contact TP is larger than the probabilities used for this parameter in ASF spread models in different jurisdictions. Our sensitivity analysis supported our assumption that indirect contact had a larger role in ASF spread in Vietnam as smaller indirect contact TP had resulted in nominal spread of the virus, contrary to what had been observed during the initial ASF outbreaks (much rapid/wider spread) in Vietnam. Although our simulation provided some guidance on the probable range of this probability, the uncertainty in this parameter estimate was still not resolved, and future field studies may help to provide better estimates.It was assumed that the ASF virus was introduced from China as the virus strain was 100% identical to China strains  to authorities. There are several reasons for this. Firstly, the symptoms of ASF are not clearly distinguishable from other diseases , particularly at the early clinical stages. Most smallholder farmers also do not fully vaccinate their pigs; therefore, it is easy for them to assume that their pigs are ill or have died from diseases other than ASF. Secondly, farmers fear the loss of all their pigs once a single pig becomes infected with ASF virus, because all pigs kept in the same pen should be depopulated. Thirdly, compensation procedures are complicated, with low compensation rates and long waiting periods, varying from several months to years depending on the availability of funds from the local authority. Lastly, farmers with infected pigs are concerned about the negative impact on their farm's reputation within their local community.This model assumed that the whole farm became infectious if one pig was infected within the herd, which is realistic because of the highly contagious nature of the virus. There is a low probability that the ASF virus would fade out without onward transmission to other pigs in the farms. Some studies have suggested that R0 of ASF virus was estimated more than 1 in other countries (Barongo et al., This study offers valuable insight into how ASF virus can be transmitted via direct and indirect contact and controlled among farms under the various simulation scenarios. Our results suggest that the enforcement of movement restriction was an effective control measure as soon as the outbreaks were reported. In addition, this study provided evidence that high standards of biosecurity can contribute to the reduction of disease spread. This simulation model can be applied to other regions or countries with modified parameters. In addition, it may be useful for assessing the cost‐effective infection control and prevention strategies in the Vietnamese context through running the ‘what‐if’ scenarios related to ASF virus transmission.The authors declare that they have no competing interests.H.S.L, K.K.T and B.W. designed research. H.S.L performed research. H.S.L and K.K.T. analysed data. H.S.L, K.K.T. VNB, TLP, ANB, TDD, VTT and B.W. wrote the paper.Ethics approval was not required for this study as any sample collection or questionnaires from animal/human has not been gathered.


The proportion of out-of-pocket spending out of inpatient and outpatient costs for UEBMI beneficiaries (24.3% and 56.1%) was much lower than that for patients under the URBMI scheme (47.9% and 76.2%). The regression analysis suggested that types of insurance, age, hospital levels, length of stay (LOS) and comorbidities were significantly correlated with the inpatient costs of patients with PD. Conclusions: The direct medical costs of patients with PD in China were high compared to the GDP per capita in Guangzhou City and different between the two evaluated types of insurance. Patients with the UEBMI scheme, of older age, with comorbidities, staying in tertiary hospitals and with longer LOS had significantly higher inpatient costs. Thus, policymakers need to reduce the gaps between the two urban insurance schemes in benefit levels, provide support for the development of a comprehensive long-term care insurance system and promote the use of telemedicine in China.Background: Parkinson’s disease (PD) is the second most common neurodegenerative disorder. This study aims to evaluate the direct medical costs of patients with PD using a large sample from an entire city and to identity the potential factors correlating with their inpatient costs in Guangzhou City, Southern China. Methods: This retrospective cross-sectional study uses data obtained from the Urban Employee-based Basic Medical Insurance (UEBMI) and the Urban Resident-based Basic Medical Insurance (URBMI) administrative claims databases in Guangzhou City from 2008 to 2012. The total sample was comprised of 2660 patients with PD. Costs were evaluated for the total sample and by types of insurance. The composition of costs was compared between the UEBMI and URBMI subgroups. The extended estimating-equations model was applied to identify the potential impact factors influencing the inpatient costs. Results: The direct medical costs per patient with PD were CNY 14,514.9 (USD 2299.4) in 2012, consisting of inpatient costs of CNY 13,551.4 and outpatient costs of CNY 963.5. The medication costs accounted for the largest part (50.3%). The inpatient costs of PD patients under the UEBMI scheme  were significantly higher than those of patients in the URBMI subgroup  ( With a rapidly aging international population, Parkinson’s disease (PD) is becoming a worldwide public health problem and an economic burden ,2. CurreAccording to the Global Burden of Disease Study, there were approximately 6.1 million people affected by PD globally in 2016 . PatientPD affects patients’ health-related quality of life and imposes a heavy economic burden on individuals, healthcare systems and societies . For exaTo enhance the level of financial protection and improve access to healthcare, China launched the Urban Employee-based Basic Medical Insurance (UEBMI) and the Urban Resident-based Basic Medical Insurance (URBMI) schemes , which wA diversity of studies has assessed the direct medical costs of PD in developed countries ,24,25,26Only a limited number of studies have evaluated the direct medical costs of PD in China ,30. YangDifferent from previous research, this study aims to evaluate the direct medical costs of patients with PD with a large sample from an entire city and to identity the potential factors correlated with their inpatient costs using two urban health insurance claims data in Guangzhou City, Southern China.This study used the UEBMI and URBMI administrative claims databases from Guangzhou City, China during the period from 2008 to 2012. Due to the administrative restrictions on accessing the social insurance claims database, this was the most recent data available for this study. The UEBMI and URBMI are two social health insurance schemes covering the population of urban employees and urban non-employed residents in Guangzhou City, with a great deal of differences in service coverages and reimbursement rates ,35. The A retrospective cross-sectional study was conducted to evaluate the direct medical costs of PD patients using a prevalence-based approach. This study collected all the reimbursement claims submitted for hospitalization care from 1 January 2008 to 31 December 2012 with the primary diagnosis of PD according to the International Classification of Diseases codes, tenth version (ICD-10), including IPD (G20) subtype and SP (G21) subtype . Their oInformation on the direct medical costs of PD patients, including both outpatient and inpatient costs, was derived from the administrative claims databases. The direct medical costs, inpatient costs and outpatient costs all contained the individual out-of-pocket (OOP) spending and the reimbursement part from the UEBMI or URBMI scheme. According to the classification of cost composition used in these two schemes, direct medical costs were divided into five groups: bed fees, medication costs, laboratory and diagnostic costs, non-medication treatment costs and other fees ,37. TablAs the theoretical framework in the field of healthcare services utilization, Andersen’s behavioral model of health services use  was deveIn this study, the dependent variable was inpatient costs per patient. The primary independent variable was the type of insurance (patients covered by the scheme of UEBMI or by the URBMI scheme). It did not have a clinically meaningful relationship with other demographic and health variables .The other covariates included gender, age, disease subtypes, hospital levels, LOS, and presence of comorbidities. Gender was dichotomized as male and female. Age was divided into four subgroups: 18–59 years old, 60–74 years old, 75–89 years old and above 90 years old. Disease subtypes were classified as IPD and SP. Hospitals were marked as three levels in China: primary hospitals, secondary hospitals and tertiary hospitals . In ChinThis study used frequency and percentage to describe categorical variables, and mean, standard deviation (SD), median and 25th–75th percentiles for continuous variables. Descriptive analysis was used to report the characteristics of patients for the entire sample and two insurance-type subgroups. We estimated the cost composition overall and by types of insurance. Due to the skewed distribution of medical costs, the Kruskal–Wallis rank-sum test was performed to identify whether there were differences in cost compositions between the two insurance schemes. We compared patients’ characteristics between the two insurance subgroups. Friedman’s two-way nonparametric analysis of variance (ANOVA) test was applied to find out the differences in patient characteristics related to inpatient medical costs by type of insurance. In this study, the extension of the generalized linear model (GLM), the extended estimating equations (EEE) model , was selEthical approval was obtained from the Institutional Review Board of the School of Public Health, Sun Yat-sen University .A total of 2660 patients diagnosed with PD were identified in this study, of whom 92.0% were covered by the UEBMI scheme and 8.0% covered by the URBMI scheme see . Males mFor all study population, the direct medical costs per patient with PD were CNY 14,514.9 , including CNY 13,551.4 (USD 2146.8) for inpatient costs and CNY 963.5 (USD 152.6) for outpatient costs see . The inpp < 0.05). However, the outpatient costs for the UEBMI scheme patients (CNY 955.3) were significantly lower than that of PD patients (CNY 1057.0) covered by the URBMI scheme (p < 0.05). The proportion of OOP spending out of inpatient and outpatient costs for UEBMI beneficiaries (24.3% and 56.1%) was much lower than those for patients under the URBMI scheme (47.9% and 76.2%). In addition, the UEBMI enrollees had significantly higher bed fees, non-medication treatment costs and other fees than the URBMI enrollees (p < 0.05).p < 0.05). Among the UEBMI subgroup, male patients incurred higher mean inpatient costs than female patients, but male patients had lower inpatient costs in the URBMI subgroup. The UEBMI patients aged over 90 years old had the highest mean inpatient costs  among all age groups, while the URBMI patients aged 75–90 had the highest inpatient costs . For the UEBMI enrollees, patients diagnosed with SP  reported lower mean inpatient costs than patients with IPD . However, the mean inpatient costs for patients with SP subtype  were higher than those for patients with IPD subtype  under the URBMI scheme. In addition, the highest mean inpatient costs were found in UEBMI patients being hospitalized in tertiary hospitals , while the highest inpatient costs were found in patients staying in secondary hospitals  for the URBMI subgroup.The patients’ characteristics related to inpatient costs for the entire sample, the UEBMI subgroup and the URBMI subgroup are shown in p < 0.05), after adjusting for other factors. Compared with the youngest age group (aged 18–59), the inpatient costs for older age groups of PD patients were CNY 1027.0 higher for those aged 60–74, CNY 1738.0 higher for those aged 75–89, and CNY 5660.5 higher for those aged 90 and above (p < 0.01). In comparison with patients staying at secondary hospitals, the costs of inpatient care were CNY 3378.7 lower for PD patients staying at primary hospitals (p < 0.01), but CNY 5523.9 higher for patients in tertiary hospitals. After controlling for other covariates, inpatient costs were CNY 6451.2 higher for longer LOS group (15 ≤ Days < 30), and CNY 29,804.3 higher for the longest LOS group (Days ≥ 30), compared to an LOS of less than 15 days (p < 0.01). In addition, PD patients with Alzheimer’s disease had significantly lower inpatient costs (CNY 1543.6) (p < 0.05), but patients with mood disorders incurred significantly higher inpatient costs (CNY 2305.5) (p < 0.01).As shown in This was a retrospective cross-sectional study conducted based on a large sample of 2660 patients with PD in Guangzhou City, Southern China. This study found that the direct medical costs per patient with PD were CNY 14,514.9 (USD 2299.4 in 2012), including CNY 13,551.4 (USD 2146.8) for inpatient costs and CNY 963.5 (USD 152.6) for outpatient costs. The medication costs accounted for the largest percentage of direct medical costs (50.3%) for PD patients. Another key finding was that the type of insurance, age, hospital levels, LOS and comorbidities (Alzheimer’s disease and mood disorders) were significantly correlated with the inpatient costs. To the best of our knowledge, this was the first study using a large sample from an entire city’s claims databases to estimate the direct medical costs of PD and compare the spending of patients with PD between two different urban health insurance schemes in China. In comparison with studies conducted in other countries, we found there were great discrepancies in cost-estimation results for PD patients, after converting all the costs into 2012 US dollars by using the purchasing power parity derived from the Organization of Economic Co-operation and Development (OECD) dataset . In thisCompared with prior studies conducted in China ,30, the In terms of cost composition, medication costs accounted for the largest percentage of direct medical costs for PD patients (50.3%) in this study. This proportion of medication costs was close to that of the 49.1% found in Germany  and 50.4Compared with prior China-based research, this was the first time differences in direct medical costs of patient with PD under the two types of health insurance schemes in China were examined. The UEBMI and URBMI were the two main social health insurance schemes administered by the Chinese government for urban residents only . The inpDisease subtype. This research was the first study from China examining the differences in direct medical costs between two disease subtypes of PD, namely IPD and SP. In the UEBMI subgroup, patients diagnosed with SP  reported lower inpatient costs than patients with IPD , while the inpatient costs for patients with SP subtype  were higher than those for patients with IPD subtype CNY 11,405.1) in the URBMI subgroup. However, after further controlling for other covariates, the regression analysis indicated that the subtype of disease was not a significant predictor of the inpatient costs of PD. For patients with IPD subtype, the mainstay of therapeutic management was symptomatic treatment with medications that increase dopamine concentrations or directly stimulate dopamine receptors .1 in the,68. PatiAge. In accordance with existing literature , this stHospital level. Consistent with previous research in China ,35,37, pComorbidities. In this study, comorbidities such as mood disorders and Alzheimer’s disease in PD patients were significantly correlated with variations in inpatient costs. Mood disorders often occurred in PD patients, and psychiatric aspects of PD were associated with numerous adverse outcomes  which maLOS. Consistent with previous studies ,35,37, iThis research had some limitations. First, our study sample only included urban PD patients covered by the two urban health insurance schemes in one city of China and excluded rural residents, from which our findings may not be generalized to the whole population in China and might cause selection bias. Additionally, the present research only evaluated the direct medical costs of patients with PD and did not examine the indirect costs induced by informal care and loss of productivity, which were not included in our insurance claims dataset. As a result, this study was likely to underestimate the total costs of PD in China. Moreover, the clinical severity of PD, which is usually assessed by the Unified Parkinson’s Disease Rating Scale, was also an important influential factor for direct medical costs . HoweverInvestigating the direct medical cost of PD and its influential factors has some practical implications. This cost-of-illness study showed real-world data for policymakers and provided guidance to the allocation of health resources and health insurance funds. The cost findings could also be used to conduct cost-effectiveness studies on different treatments for PD patients. Due to budget constraints and resource scarcity in all countries, research analyzing costs, their drivers and the size of costs per patient are important as they provide knowledge to decision-makers at all levels of health care.The direct medical costs of patients with PD in China were high compared to the GDP per capita in Guangzhou City and different between the two evaluated types of insurance. Patients with the UEBMI scheme, of older age, with comorbidities, staying in tertiary hospitals and with longer LOS had significantly higher inpatient costs. Thus, policymakers need to reduce the gaps between the two urban insurance schemes in benefit levels, provide support for the development of a comprehensive long-term care insurance system and promote the use of telemedicine in China to reduce the economic burden attributable to PD.


Kobuvirus of the family Picornaviridae; it is a single-stranded positive-sense RNA virus without an envelope. AiV causes acute gastroenteritis, abdominal pain, nausea, vomiting, and fever. Low incidence and high seroprevalence of AiV infections have been reported in several regions of the world; however, little was known on the prevalence of AiV infections in Taiwan. This study described the first two cases of AiV infection and analyzed AiV seroprevalence in Taiwan. A total of 700 sera were collected from a single hospital in southern Taiwan. The neutralization assay was employed to assess AiV neutralization antibodies in the serum. The test identified 48 positive cases, with a seroprevalence of 6.86%. Results also showed a gradual increase in AiV seroprevalence rate with age. Compared with other countries, Taiwan had a relatively low AiV seroprevalence, suggesting a low incidence of or sporadic AiV infections.Aichi virus (AiV) belongs to the genus  Kobuvirus, Aichivirus A genus in the Picornaviridae family [Kobuvirus species (Aichivirus A–F) within the Kobuvirus genus have been classified according to the 2019 Release of the ICTV , and three genotypes  have been identified within the human Aichi virus [Human Aichi virus (AiV) is a single-stranded, positive-sense RNA virus, a member of the e family . The vire family . The AiVe family ,4. Sequehi virus ,6. AiV causes acute gastroenteritis, with symptoms including diarrhea, abdominal pain, nausea, vomiting, and fever. AiV gastroenteritis is considered a viral foodborne disease through the fecal–oral route; however, subclinical infections of AiV pathogenesis are common ,7,8. TheAiV shows an enterovirus-like cytopathic effect (CPE) in cell culture, and the appearance of cell shrinkage is almost the same as that of enterovirus in morphology. There is no identification reagent available for AiV in clinical practice. Instead, the enterovirus immunofluorescence antibody kit is used for identification in general clinical laboratories; hence, AiV is often judged as an NPEV (nonpolio enterovirus) and regarded as an unclassified enteric virus, causing the isolation rate to be underestimated or neglected. Different countries reported varied incidences of AiV infection, such as 5% in Northwestern Spain, 0.5% in Finland, 2% in Sweden, 0.43% in Denmark, and 1.8% in China ,12,13,14In Taiwan, the first AiV cases were diagnosed in 2010. After that, AiV has not been isolated. Investigation data on gastroenteritis cluster infections obtained by the Taiwan Central Disease Control Agency in the past ten years contained no AiV cases. The isolation rate of this virus in Taiwan seems to indicate that it is sporadic. However, AiV did appear in Taiwan. Therefore, to understand whether AiV has been prevalent in Taiwan in the past, a survey on serum antibodies was conducted to analyze the distribution of antibody titers in different age groups and the AiV infection status in Taiwan.E. coli: >100,000 CFU/mL in urine culture. In Taiwan, the first two AiV were isolated from two newborns aged 3 and 5 days in 2010 at Kaohsiung Veterans General Hospital. The patients were referred by the postpartum care confinement center and were initially diagnosed with acute pharyngitis, acute gastroenteritis, and herpangina. Their clinical symptoms included vomit, vesicle over the hard palate, and irritable crying. The younger patient excreted sticky stool. The laboratory data of the 3-day-old newborn showed fecal occult blood, coxsackievirus (Cox) B1-B6 IgG immunofluorescence test: ≥1000, negative blood and urine bacterial cultures, negative throat virus culture, and positive rectal virus culture; while those of the older patient showed Cox B1, B6 test: ≥1000, Cox B3-B5 IgG immunofluorescence test: 1:320 and Enterovirus typing was negative. RT-PCR analysis of Enterovirus CODEHOP (COnsensus-DEgenerate Hybrid Oligonucleotide Primers) and Parechovirus were negative. Primary determination suggested no enteroviruses in the specimens, which were then subjected to Aichi virus RT-PCR analysis with the three primer sets: VP0 region, 708 bp; 2C to 3A-3B junction, 557 bp; and 3C to 3D, 519 bp, and they all showed positive results.The virus culture analysis yielded a negative throat virus culture and a positive rectal virus culture in Vero cells showing enterovirus-like CPE. CPE was not observed in other cell types used in the laboratory, such as human embryonal lung fibroblast (MRC-5), human rhabdosarcoma (RD), human lung adenocarcinoma (A549), rhesus monkey kidney (LLC-MK2), and Madin–Darby canine kidney (MDCK) cells. In those Vero cells, enterovirus integrated antibody (PanEV) fluorescence staining was positive, while immunofluorescence staining of Three genotypes of AiV (types A to C) have been identified. Epidemiological studies revealed a certain geographical distribution of AiV genotypes, indicating circulation of genotypes A and B in different Asian and European countries .Phylogenetic analysis on the two AiV isolates (990126-32 and 990127-18) of Taiwan was performed. Reference strains were obtained from GenBank. From the comparison with 518 bp sequences in the 3C/3D region, these two isolates were confirmed to be AiV type A. The two AiV isolates were 100% identical according to the sequencing results .p < 0.001). The correction test showed a similar result . The largest portion of the 48 positive cases was in the group of >50–60 years (31%)  B.This study surveyed the seroprevalence of AiV in southern Taiwan using the neutralization assay. The overall prevalence was 6.86% and increased with age but showed differences in age distribution. The seroprevalence rate was 2.93% among those aged under 40 but rose to 10.58% among those aged over 40. These results show a low AiV prevalence in Taiwan compared to that of other regions.According to the earliest report, the average positive AiV seroprevalence rate in Japan was 55.2% (460/833), and the positive rate increased with age, from 7.2% (9/125) among those aged >0.7–4 years to 87.5% (70/80) of those aged over 55 years. Almost 80% of all healthy adults acquired the neutralizing antibody by the age of 40 . A surveWhile Japan and Germany used the neutralization assay to survey AiV seroprevalence ,20, the The first identified AiV cases in Taiwan were among infants; it would thus be of interest to understand the prevalence rate in young children. However, in this study, only 40 sera were collected from children under 5 years old, and none of them presented the AiV neutralization antibody. This prevalence rate was much lower than that obtained in Japan, Germany, and France ,17,20. I10 reduction on plaque assay [Foodborne disease is a global health problem, AiV is a foodborne pathogen and is closely related to diet and environmental hygiene . Foodborue assay . A natioue assay . AiV hasue assay ,22.Ever since AiV was isolated from the rectal swabs of two infants, there has been no other AiV isolation reported in Taiwan. Although there is no large-scale sudden infection in Taiwan, AiV can still appear sporadically. From the perspective of epidemic prevention, we cannot abandon AiV identification.2. The amplified virus was tittered by the assay of fifty-percent tissue culture infective dose (TCID50)/mL [2.AiV strain kvgh99012632/2010 (accession no. JX564249) [Enterovirus diagnosis of the Virology Group, Department of Microbiology, Kaohsiung Veterans General Hospital [Enterovirus group-specific immunofluorescence assay was performed with pan-Enterovirus antibody  according to the manufacturer’s protocol. Virus identification involved the protocol for Hospital . In brieThe RT-seminested PCR (RT-snPCR) analysis method and primer design of all enterovirus serotypes were adapted from a previous report .Parechovirus RNA by RT-PCR according to our previous study [Clinical specimens were tested for us study . PCR perus study . The PCRThe procedure of RT-PCR analysis of Aichi virus in specimens was adapted from the previous report . The PCRSerum IgG antibodies against coxsackievirus type A  and type B  were detected by indirect immunofluorescence assay on biochips . Specimens were subjected to bacteria culture for identification using the Vitek 2 system .https://www.megasoftware.net. accessed on 24 April 2021). There was a total of 518 positions in the final dataset. Codon positions included were 1st + 2nd + 3rd + noncoding. All positions with less than 60% site coverage were eliminated, i.e., fewer than 40% alignment gaps, missing data, and ambiguous bases were allowed at any position . Because AiV is a globally distributed virus, 27 AiV isolates from different regions were chosen for the evolutionary analysis. The sequences of these reference strains shown in The PCR products of AiV 3C/3D region were purified using the GFX™ PCR DNA and Gel Band Purification Kit  according to the manufacturer’s instructions. The purified DNA fragments were sequenced and analyzed. Phylogenetic analysis of AiV was conducted according to the resulting sequences’ alignment in 3C/3D region using the maximum-likelihood method (Kimura 2-parameter model) with MEGA X software . Fifty microliters of diluted serum was mixed with 50 μL of virus solution (100 TCID50), and then placed in a 96-well flat-bottomed culture plate. The plate was incubated at 35 °C, 5% COp value less than 0.05 indicates statistical significance.Statistical analysis among the age groups were performed by chi-square for trend test and Pearson’s correlation test using the GraphPad Prism software . A


Flavivirus), akin to other viruses, are modulated by high mutation rates and selective forces to adapt their codon usage to that of their hosts. However, a major challenge is the identification of potential hosts for novel viruses. Usually, potential hosts of emerging zoonotic viruses are identified after several confirmed cases. This is inefficient for deterring future outbreaks. In this paper, we introduce an algorithm to identify the host range of a virus from its raw genome sequences. The proposed strategy relies on comparing codon usage frequencies across viruses and hosts, by means of a normalized Codon Adaptation Index (CAI). We have tested our algorithm on 94 flaviviruses and 16 potential hosts. This novel method is able to distinguish between arthropod and vertebrate hosts for several flaviviruses with high values of accuracy (virus group 91.9% and host type 86.1%) and specificity (virus group 94.9% and host type 79.6%), in comparison to empirical observations. Overall, this algorithm may be useful as a complementary tool to current phylogenetic methods in monitoring current and future viral outbreaks by understanding host–virus relationships.Early characterization of emerging viruses is essential to control their spread, such as the Zika Virus outbreak in 2014. Among other non-viral factors, host information is essential for the surveillance and control of virus spread. Flaviviruses (genus  Recent viral pandemics have shown that rapid characterization of the virus is essential during the development of an outbreak ,2,3,4. AFlavivirus and 16 potential hosts. Several flaviviruses are major human pathogens, with potential host ranges from vertebrates to arthropods [In this article, we introduce an unsupervised algorithm to identify putative virus host ranges based on only genome sequence information. The proposed methodology has been tested in 94 viruses of genus thropods . Flavivithropods  flavivirthropods , also knthropods . Flavivithropods .http://ppuigbo.me/programs/CAIcal/nCAI (accessed on 13 May 2021), to calculate nCAI values.Several articles suggest that highly similar codon usage frequencies between viruses and hosts are indicative of a high virus–host adaptation level . Thus, tThe optimal host identification algorithm  consistsThis yields the normalized CAI (nCAI) value, from which the optimal and likely hosts can be inferred depending on how similar the codon usage of a virus is to the codon usage of its host organisms. The nCAI values range between −∞ and +∞, and the optimal value is 1.0, indicating identical codon usage between the virus and host and therefore perfect adaptation to the host. Values above and below 1.0 would indicate over- and underoptimization, respectively, and thus suboptimal adaptation to a host.http://ppuigbo.me/programs/CAIcal/nCAI, accessed on 13 May 2021). The server requires two sets of inputs: complete DNA or RNA CDSs of the viruses of interest in FASTA format and the codon usage tables of the potential host animals in the format used by the Codon Usage Database [The nCAI calculations can be performed with the CAIcal tool in a dedicated web server, written in PHP, that works on any web browser (n = 94) were chosen according to phylogenetic studies [Available flavivirus CDSs and their respective protein sequences were obtained from the RefSeq  and GenB studies ,25,26 an studies   and Sus scrofa (2953).In this study, a vector is defined as an organism capable of transmitting a virus to another type of organism. This definition does not take into account whether the virus is virulent within a vector, i.e., there is no differentiation between a vector and a vector-host. A host, on the other hand, is an organism in which the virus primarily replicates, and it does not directly transmit the virus to another organism of the same type. The host organisms (N = 16) for this study were chosen based on information primarily provided by the Virus–Host Database , which iThe %GC and relative synonymous codon usage (RSCU) values were calculated from the CDSs of the flaviviruses. The RSCU describes the preference bias for a codon to be used to encode an amino acid . This caFirst, to assess whether a codon usage methodology could distinguish subgroups within a viral species, we performed nCAI-CA analysis for all the available CDSs of DENV, WNV, JEV, and ZKV, which numbered 4865, 297, 1619 and 494, respectively. Each viral subgroup formed a distinct cluster based on relative synonymous codon usage  and GC cAedes mosquitoes), and TBFVs (Ixodes scapularis)  and in many cases the preferred mosquito vector-host is debatable [Aedes; however, a higher genomic adaptation does not imply that Aedes is currently the most common host-vector for all MBFV, as additional factors should be considered. Moreover, our algorithm mostly rules out Anopheles gambiae as the main host-vector, in agreement with the literature [Next, we used the nCAI-CA algorithm  to identpularis) . The partebrates . The CA tebrates ; howeverck hosts . The nCAck hosts . All theck hosts . Our anaebatable ,36,37,38terature , althougterature ,41. OverMyotis brandtii, M. davidii, Mus musculus, Bos taurus, and Homo sapiens) and Aedes mosquitoes . Unlikely hosts due to low adaptation (nCAI < 0.9) include C. quinquefasciatus, A. gambiae, I. scapularis and S. scrofa. These results are in accordance with previous studies and observations, e.g., most MBFVs have a reproductive cycle that includes Aedes (host-vector) or Culex (vector) mosquitoes and a primary mammalian host . In general, flavivirus codon usage is overoptimized (nCAI > 1.1) for birds , amphibians (Xenopus laevis) and reptiles (Alligator mississippiensis).The heat map of nCAI values for all flaviviruses shows common adaptation patterns . Likely ian host . Based oDespite the high values of specificity and accuracy produced by the nCAI-CA , there aNevertheless, further empirical investigations are necessary to determine reliable confidence intervals for nCAI. Host determination may be uncertain if viruses display approximately equal optimizations for different host types; for example, although dhIOFV codon usage is optimized for both vertebrate and mosquito hosts, they are insect-specific . AlthougIn conclusion, this novel algorithm provides a fast and proactive method to assess the potential host ranges and the risk of zoonotic host shift for new and emerging viruses. In flaviviruses, this method distinguishes between arthropod and vertebrate hosts with high accuracy. However, it might produce ambivalent results for viruses undergoing host shifts. Overall, this nCAI-based algorithm may be used as a complement to current phylogenetic methods to monitor current and future outbreaks.


Pigs play an important role in agriculture and biomedicine. The globally developing swine industry must address the challenges presented by swine-origin viruses, including ASFV (African swine fever virus), PRRSV (porcine reproductive and respiratory syndrome virus), PEDV (porcine epidemic diarrhea virus), PRV (pseudorabies virus), CSFV , TGEV (transmissible gastroenteritis virus), et al. Despite sustained efforts by many government authorities, these viruses are still widespread. Currently, gene-editing technology has been successfully used to generate antiviral pigs, which offers the possibility for increasing animal disease tolerance and improving animal economic traits in the future. Here, we summarized the current advance in knowledge regarding the host factors in virus infection and the current status of genetically modified pigs that are resistant to virus infection in the world. There has not been any report on PEDV-resistant pigs, ASFV-resistant pigs, and PRV-resistant pigs owing to the poor understanding of the key host factors in virus infection. Furthermore, we summarized the remaining problems in producing virus-resistant pigs, and proposed several potential methods to solve them. Using genome-wide CRISPR/Cas9 library screening to explore the key host receptors in virus infection may be a feasible method. At the same time, exploring the key amino acids of host factors in virus infection with library screening based on ABEs and CBEs (Bes) may provide creative insight into producing antiviral pigs in the future. Pigs, as one of the most important types of livestock, play an indispensable role in agriculture. They share similar genetic, physiological, and anatomical features and body sizes with humans, and are regarded as important candidates for organ donors for xenotransplantation. Furthermore, pigs are an important model organism for insights into the mechanisms of human disease ,2,3. HenIn recent decades, vaccines against porcine viral diseases have been developed to enhance the adaptive immunity of hosts ,5. HowevPestivirus genus within the Flaviviridae family, and is an enveloped and positive-sense RNA virus [Classical swine fever (CSF), one of the most highly contagious swine diseases, characterized by high fever and high mortality, is caused by the classical swine fever virus, and leads to tremendous economic losses to the swine industry throughout the world ,12. The NA virus . AccordiNA virus ,15. It wNA virus ,17. There are 38 CSFV-free regions in the world according to the World Organization for Animal Health (OIE), which are mainly located in North America, the European Union, Oceania, Asia, Eastern Europe, and part of Africa. Furthermore, it was reported that CSFV recurred in a supposedly CSFV-free country, Japan, due to a reservoir of CSFV in wild boars in that country . Thereforns, and other E2 subunit DIVA vaccines [Currently, live attenuated vaccines are the most widely used strategy to control CSFV in the world. Classical CSF vaccines, such as c-strain, GPE-strain, LPC-strain, and LK-VNIVViM strain, provided robust protection from CSFV infection , though vaccines . Despite consistent efforts by many government authorities, it is still difficult to stamp out CSFV in infected areas and re-emerging areas. The live attenuated vaccines and the marker DIVA vaccines result in subclinical infection and immunosuppression, which makes it even harder to eliminate CSFV. As a result, another strategy is urgently needed to control the virus in pigs.A number of host factors were identified to have participated in the process of CSFV replication and pathogenesis . SeveralRosa26 site-specific integration pigs of RSAD2 were produced in our lab [Several host factors with anti-CSFV activity were utilized to generate anti-CSFV pigs. In 2016, our group generated pigs that over-express MxA . Later,  our lab . All theRosa26 sites and porcine miR-17-92 clusters in our lab [In addition, RNAi has been regarded by virologists as a promising way to suppress virus infection. To date, there have been several RNAi-based studies on CSFV suppression in vitro, and these studies have indicated that the development of shRNA-TG pigs that are resistant to CSFV may be possible . Anti-CS our lab ,48. The Asfarviridae family, and contains a linear double-strand DNA. The genome of ASFV is about 170 kb-190 kb and encodes about 151–167 ORFs [African swine fever (ASF), which is caused by the African swine fever virus (ASFV), is a hemorrhagic and infectious disease listed by the OIE, causing enormous economic losses each year . ASFV be167 ORFs . Current+ monocyte subpopulations, but not CD163- monocyte subpopulations. Blocking the membrane receptor of primary alveolar macrophages with mAbs 2A10 and 4E9 inhibits ASFV infection, suggesting that CD163 acts as an important membrane receptor in the process of ASFV infection [Like porcine reproductive and respiratory syndrome virus (PRRSV), ASFV replicates predominantly in porcine alveolar macrophages (PAMs). Previous studies showed that ASFV mainly replicates in specific cytoplasmic sites, which have been referred to as viral factories, albeit a lot of ASFV DNA synthesis takes place in the nucleus in the early stages of infection. Despite decades of efforts by virologists, the key membrane receptor in the process of ASFV entry has not been identified yet. Early studies implied that ASFV entry into host cells is through receptor-mediated endocytosis ,52. Givenfection . Howevernfection . So far,nfection ,57,58. 6.0 TCID50. Five out of six LS-2 pigs were found to have low viremia at 9 dpi, while the common domestic pigs were found to have fever and viremia at 3 dpi. In the end, five out of six LS-2 pigs survived, and all the common domestic pigs died at less than 10 dpi [It was announced by Chinese scientists in 2020 that Lansibai-2 pigs (LS-2), which were bred by China Shandong Landsee Genetics Co., Ltd., were resistant to ASFV. Previous results implied that LS-2 pigs showed significant ASFV resistance following oral challenge with an SY18 strain at the dosage of 10n 10 dpi . More attention should be paid to exploring key receptors with CRISPR/Cas9 library screening technology in the future. Additionally, generating anti-ASFV pigs with functionally annotated gene targets may be a promising strategy to control ASFV.Arteriviridae family in the order Nidovirales [Porcine reproductive and respiratory syndrome, which is caused by the porcine reproductive and respiratory syndrome virus (PRRSV), is an economically significant contagious disease . PRRSV iovirales . The genovirales . In Chinovirales . Recombiovirales .Vaccination has been the major strategy to prevent PRRSV infection in the past two decades. Multiple modified live and inactivated vaccines against both types of PRRSV have been developed to control PRRSV transmission. However, these vaccines failed to provide sustainable protection owing to the high recombination and variation of PPRSV, which delayed the neutralization antibody response and led to antibody-dependent enhancement (ADE) to counter host immunity ,66. AccoNumerous studies have shown that PRRSV entry is mediated by various cell receptors, such as sialoadhesin Sn, CD169) ,68, CD15 ,68, CD1Besides these PRRSV entry blockers, some host factors that act in PRRSV infection were found, too. . Jiang eIn 2013, Li et al.  reportedAccordingly, CD163-gene-edited pigs with great PRRSV resistance exhibit fine prospects for controlling viral infection and also lay the foundation for controlling others virus. Nevertheless, there are still some concerns that need to be addressed. Firstly, CD163 plays an important role in vivo. It remains unknown whether knocking out CD163 SRCR5 affects the function of CD163. Additionally, it is still an issue of concern as to whether substituting CD163 SRCR5 with human CD163 L1 SRCR10 renders humans sensitive to PRRSV. Hence, exploring the key amino acids that function in the process of PRRSV infection in the CD163 may be another approach to address these concerns. Porcine enteric coronaviruses (PECs) cause high mortality and morbidity in newborn piglets. Such viruses include porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), swine acute diarrhea syndrome coronavirus (SADS-CoV), and porcine delta coronavirus (PDCoV) . It was Porcine aminopeptidase-N (pAPN), which is mainly expressed on the surface of enterocytes, was first reported to serve as a receptor of TGEV in 1992 . Later, pRosa26 locus of PK-15 cells made it resistant to CSFV and PRV, but the resistance of pRSAD2 KI pigs to CSFV and PRV infections was undetermined. At the same time, it was reported that human CCCH-type zinc finger proteins containing the 11A protein (ZC3H11A) are essential for the replication of multiple nuclear-replicating viruses  in human cells [Similarly, Ouyang et al.  producedan cells , which sAt present, there are many pigs resistant to CSFV, PRRSV, and TGEV being produced, which suggested the enormous potential of CRISPR/Cas9-based gene-editing technology for shortening the breeding cycle and enhancing disease tolerance in pigs. However, there have been no reports of pigs resistant to PEDV, ASFV, and PRV owing to the lack of understanding of the key receptors in viral infection. In the future, genome-wide CRISPR/Cas9 library screening technology may be an ideal method to explore the key receptors in PEDV, ASFV, and PRV infection. Gene-edited pigs showed remarkable virus resistance, indicating a fine prospect for controlling virus infection. Nevertheless, there are still some concerns emerging. First, it was reported that CRISPR/Cas9 may induce megabase-scale chromosomal truncations in cell lines and primary cells with P53-dependent mechanisms . Some ho