Heart failure (HF) imposes a heavy economic burden on patients, their families, and society as a whole. Therefore, it is crucial to quantify the impact and dimensions of the disease in order to prioritize and allocate resources effectively.This study utilized a prevalence-based, bottom-up, and incidence-based Markov model to assess the cost of illness. A total of 502 HF patients (classes I–IV) were recruited from Madani Hospital in Tabriz between May and October 2022. Patients were followed up every two months for a minimum of two and a maximum of six months using a person-month measurement approach. The perspective of the study was societal, and both direct and indirect costs were estimated. Indirect costs were calculated using the Human Capital (HC) method. A two-part regression model, consisting of the Generalized Linear Model (GLM) and Probit model, was used to analyze the relationship between HF costs and clinical and demographic variables.The total cost per patient in one year was 261,409,854.9 Tomans . Of this amount, 207,147,805.8 Tomans  (79%) were indirect costs, while 54,262,049.09 Tomans  (21%) were direct costs. The mean lifetime cost was 2,173,961,178 Tomans. Premature death accounted for the highest share of lifetime costs (48%), while class III HF had the lowest share (2%). Gender, having basic insurance, and disease class significantly influenced the costs of HF, while comorbidity and age did not have a significant impact. The predicted amount closely matched the observed amount, indicating good predictive power.This study revealed that HF places a significant economic burden on patients in terms of both direct and indirect costs. The substantial contribution of indirect costs, which reflect the impact of the disease on other sectors of the economy, highlights the importance of unpaid work. Given the significant variation in HF costs among assessed variables, social and financial support systems should consider these variations to provide efficient and fair support to HF patients. Heart failure (HF) is a complex syndrome where the heart fails to properly pump blood, leading to a lack of oxygen and nutrients for the tissues. It is characterized by symptoms such as shortness of breath, fatigue, chest pain, and fluid retention , 2. HF iGaining knowledge and information about the level and dimensions of the impacts of diseases is necessary for planning and prioritizing resource allocation for disease control –25. UndeThe present study utilized a prevalence-based and incidence-based cost-of-illness approach. The prevalence-based analysis was conducted using a bottom-up approach, while the incidence-based analysis employed Markov modeling. Data for this study were collected from May to October 2022. A total of 502 heart failure patients with diagnostic codes  according to the ICD-10 and classified as classes I, II, III, or IV based on the New York Heart Association (NYHA) classification system were selected using a systematic sampling method. These patients were selected among those referred to Shahid Madani Hospital of Tabriz, with ethics approval IR.TBZMED.REC1401.361. The sample consisted of patients who had visited the hospital as inpatients or outpatients within the past two years. Shahid Madani Hospital is a specialized public center for heart patients in the northwest of Iran. Patients were followed up every two months for a minimum of two months and a maximum of six months using a person-month measure. This approach was applied to all types of included costs, except for inpatient costs. For inpatient costs, we asked whether patients had been admitted to the hospital in the last year due to their HF disease and this hospitalization rate was applied for one-year period. Additionally, we calculated the average cost of admission per HF patient using hospital bills.The final outcome of the cost analysis was calculated as an annual cost per patient. To achieve this, the monthly cost of each patient (using person-month average) was multiplied by 12 to obtain the annual cost. This study estimates the costs of heart failure from a societal perspective, considering both direct and indirect costs. Three stages of cost identification, measurement, and valuation were implemented to calculate these costs. The value of resources used was determined based on private sector tariffs, and indirect costs were calculated using the human capital approach, which measures productivity lost due to absenteeism, premature death, presenteeism (low productivity), unpaid work, and job change.Data for direct costs were collected using the Medical Consumption Questionnaire and data for indirect costs were collected using the Productivity Costs Questionnaire , both ofIn addition to estimating annual costs, this study also estimated the costs of heart failure over a patient’s lifespan using a modeling approach. A Markov model with six states was developed for this purpose. The states of illness were defined based on the NYHA classification method and included NYHA1, NYHA2, NYHA3, NYHA4, as well as two death states (death due to other causes and death due to heart failure). The time horizon of the model was set at 25 years with one-year cycles. Transition probabilities between different states were estimated based on the effectiveness of common ACE treatments using relevant studies . Mean coThe study aims to analyze the economic costs of heart failure by considering the impact of clinical and demographic variables. To address the challenges of asymmetry of cost data, zero-cost, and censored data, a two-part regression model was used. This model includes two parts: the Probit model for discrete variable regression estimation and the Generalized Linear Model (GLM) for continuous variable regression estimation. The variables examined in the regression model include age, insurance status (having or not having basic insurance), class of disease, comorbidity, and gender. The goodness of fit of the model was assessed using the AIC index, and the regression coefficients were examined at a 95% level of significance. The regression analyses were performed in Stata_16 Software.The study utilized the incidence-based method to calculate the average costs for heart failure patients. This approach involved the use of a simulated cohort through the Markov model. The mean estimated cost for a heart failure patient was found to be 2,173,961,178 Tomans. The costs associated with each Markov model state, such as NYHA classes and premature death, were also determined. Specifically, the cost for class I was estimated at 471,146,883 Tomans, class II at 88,460,400 Tomans, class III at 51,001,097 Tomans, class IV at 517,348,089 Tomans, and premature death due to heart failure at 1,046,004,708 Tomans.Heart failure is a significant health problem that imposes both health and financial burdens on patients and society. The results of this study provide a comprehensive analysis of the burden of heart failure across various dimensions. The study adopts a prevalence-based approach from a social perspective to estimate the average annual cost of heart failure. Unlike similar studies, this research takes into account both direct and indirect costs, including unpaid work costs. Moreover, in addition to the prevalence-based approach, the study also employs an incidence-based modeling method to estimate the costs of heart failure over the patient’s lifetime. This approach is less commonly used in studies focusing on this population. Furthermore, this study investigates the relationship between influential variables and their impact on the occurrence and changes in costs. An appropriate regression model is utilized to analyze these variables, and the subsequent findings for each topic are discussed in detail.The prevalence-based results reveal that the total cost per heart failure patient in one year is 261,409,854.9 Tomans. Indirect costs account for the majority of these costs, with unpaid work costs being the largest component. Direct costs, including direct medical and non-medical costs, make up a smaller portion of the total costs. The direct medical costs mainly consist of pharmaceutical, diagnostic, and treatment expenses. No study has been conducted on the economic burden of heart failure in the context of Iran using a similar design. However, several studies have been carried out on the overall burden of cardiovascular diseases. These studies did not specifically estimate the burden of heart failure and are not comparable to the current study in terms of methodological design, cost considerations, and reported outcomes. In one of those studies .Henkel Li et al. in South Korea estimated the prevalence and economic burden of heart failure disease. The mean cost per patient in 2014 was estimated at 1,414.0 dollars . It seems difficult to compare the total costs owing to the heterogeneity of the influential parameters. Medical costs have the largest share of the national burden (54.8 + 13.7% = 68.5%), followed by caregiver costs (13.2%), presenteeism costs due to premature death (10.8%), complications (4.2%), and transportation costs (3.4%), respectively . Since tBlazej estimated the indirect costs and financial outcomes of heart failure in Poland from 2012 to 2015. The results revealed that premature death accounted for 59.3%–63.4% of the total costs, presenteeism due to disability accounted for 11.1%–14.2% of the total costs, patient absenteeism accounted for 3.3%–4.0% of the total costs, and caregivers’ absenteeism accounted for 0.02% of the total costs . Since tMost of the articles investigated the economic burden of heart failure disease only in terms of limited costs such as direct or indirect costs. However, the present study considered all aspects of annual indirect costs per patient. One of the comprehensive characteristics of this study is considering the costs caused by unpaid work, which includes 64% of the total annual costs. The reason for high cost of this item is that heart failure affects the performance and ability of patients and increases their dependence on others. Also, since the majority of the heart failure patients were housewives or retired people, whose role in producing market value was low, unpaid activities accounted for a significant proportion of the total indirect cost. Generally, heart failure significantly affects the quality of life, performance, and productivity of people, which leads to the disability of patients and thus increases indirect costs.The lifetime costs of heart failure patients were calculated using the incidence-based method in this study, along with the annual cost of the disease. Unlike some other studies that use real data and patient follow-up, this study estimated costs through modeling (simulation) using a Markov model with 6 health states. The average estimated lifetime cost for a patient was 2,173,961,178 Tomans. The model also considered lifetime costs associated with each Markov state, including NYHA classes and premature death due to heart failure. The highest proportion of costs was attributed to premature death due to heart failure, accounting for 48% of total costs, followed by class IV with 24%, class I with 22%, class II with 4%, and class III with 2%. While there are no studies specifically analyzing the costs of heart failure using modeling, some studies have examined the cost-effectiveness of interventions using a Markov model. The costs investigated in these studies can be considered consistent with the findings of this study.In a study conducted by Yue Wu et al. in China, sacubitril/valsartan was compared to enalapril in heart failure patients. Using a Markov model, the study estimated the costs of the disease over a 10-year period. The mean cost of routine treatment  was estimated at 4,014 US dollars over 10 years . HoweverSimilarly, Gaziano et al. used a two-state Markov model to estimate the medical costs of heart failure patients over 30 years. The total cost for this period was estimated at 83,303 US dollars . Again, Another study by Lorenzo Pradelli et al. examined the cost-effectiveness of using valsartan compared to the standard treatment method in Italy over a 10-year period. The cost of standard treatment was 87,899 euros . HoweverThe present study utilized a two-part regression model. The Probit Model was used to investigate the probability of cost occurrence, in relation to examined clinical and socio-demographic variables, while the Generalized Linear Model (GLM) was used to evaluate the relationship of those variables with the level of cost variations. The results showed that gender, having basic insurance, and disease class significantly influenced the cost of the disease. On the other hand, comorbidity and age did not have a significant impact on costs. The model also successfully predicted the amount of cost, with the predicted amount being almost equal to 100% of the observed amount, indicating good predictive power.Several studies have also examined the effects of these variables on the cost of heart failure. For example, a study by Teerapat et al. in several Asian countries found that age did not significantly affect costs in Malaysia and Thailand, which aligns with the findings of the present study. The lack of significant impact may be attributed to the limited variance in age range, as most participants were older individuals. Additionally, these studies reported that gender did not significantly affect costs. However, the present study revealed that gender does influence cost occurrence. The significant impact of gender on costs can be attributed to the non-inclusion of the monetary value of women’s housework, which has a significant influence on indirect costs. In Thailand, the role of disease class on costs showed a significant difference between class IV, class I, and class II, but no significant difference between class III and class IV. The results of our study were consistent with those of a study in Thailand in terms of the variation in the costs of disease classes. However, a study in Malaysia did not find a statistically significant difference in costs between disease classes, which may be due to the use of similar treatment protocols in the variables. Comorbidity did not have a statistically significant effect on costs in the studies conducted in Malaysia and Thailand, which is consistent with the findings of our study .As expected, the presence or absence of basic insurance significantly affected the costs of the disease, with uninsured individuals experiencing lower costs (utilization). While there is limited information on the role of insurance in the costs of heart failure specifically, other studies have investigated its impact on costs in other diseases and reported statistically significant effects , 42.Heart failure is a common disease that has significant impacts on patients, including functional, physical, and psychological problems. This study found that heart failure imposes a substantial economic burden on patients, both in terms of direct costs and indirect costs. The indirect costs, which reflect the effects of the disease on other economic sectors, were found to be significant, especially when considering the value of unpaid work. Additionally, this study estimated the lifetime costs of heart failure using both prevalence-based and incidence-based methods, providing valuable information for cost-benefit analyses and calculating return on investment.The present study employed econometric methods to investigate the relationships between clinical and socio-demographic variables and their impact on heart failure costs. The results of this analysis provided significant insights into the factors influencing the costs of heart failure. Given the substantial economic burden of this disease on patients, it is crucial to develop support and insurance mechanisms that cover treatment costs and support lost productivity. While the government in Iran has made efforts to provide comprehensive coverage for individuals, there are still some individuals who lack insurance. This lack of insurance directly impacts the costs associated with heart failure, as it creates barriers to accessing timely and appropriate medical services, leading to worsened conditions and increased direct and indirect costs in the future. The Probit regression results from this study also highlight the statistically significant effect of insurance on cost occurrence. However, it is important to note that insurance only covers a portion of medical expenses, leaving significant indirect costs uncovered, which greatly impact individuals’ well-being and living.Despite being a comprehensive cost-of-illness study on heart failure, there are some limitations that should be considered when interpreting the findings. Firstly, the sample for this study was limited to the northwest region of Iran (East Azarbaijan province) and may not be representative of the entire country. Although treatment tariffs and protocols are generally consistent across Iran, generalizing the results should be done with caution. Secondly, the study sample only included patients from public hospitals, and it would have been beneficial to include patients from private hospitals as well. Although the tariffs applied for services were proportional to the prices in the private sector and were analyzed accordingly, including private sector patients would have provided a more comprehensive perspective. Thirdly, the follow-up period in this study ranged from 2 to 6 months, which may not fully capture the one-year timeframe that was generalized. Ideally, patients would have been followed up for a full year, but time and resource constraints limited this possibility. Lastly, the incidence-based method used simulation and modeling techniques, which may have limitations compared to using real data and patient follow-up.


Clinical data on retaining extensively damaged teeth using forced orthodontic extrusion followed by restorative rehabilitation are scarce, and economic evaluations are basically absent. A health economic evaluation of this method was performed based on a clinical study.In a convenience sample of individuals recruited from routine care, extensively damaged teeth were orthodontically extruded prior to restoration. Patients were followed up for up to 6 years. The health outcome was tooth retention time. Direct medical, non-medical, and indirect initial and follow-up costs were estimated using the private payer’s perspective in German healthcare. Association of initial direct medical treatment costs and cofounding variables was analyzed using generalized linear models. Success and survival were secondary outcomes.A total of 35 teeth in 30 patients were followed over a mean ± SD of 49 ± 19 months. Five patients (14%) dropped out during that period. Median initial costs were 1941€ (range: 1284–4392€), median costs for follow-up appointments were 215€ (range: 0–5812€), and median total costs were 2284€ (range: 1453 to 7109€). Endodontic re-treatment and placement of a post had a significant impact on total costs. Three teeth had to be extracted and in three patients orthodontic relapse was observed. The survival and success rates were 91% and 83%, respectively.Within the limitations of this clinical study, total treatment costs for orthodontic extrusion and subsequent restoration of extensively damaged teeth were considerable. Costs were by large generated initially; endodontic and post-endodontic therapies were main drivers. Costs for retreatments due to complications were limited, as only few complications arose.The restoration of extensively damaged teeth after forced orthodontic extrusion comes with considerable initial treatment costs, but low follow-up costs. Overall and over the observational period and within German healthcare, costs are below those for tooth replacement using implant-supported crowns.ClinicalTrials.gov Identifier: DRK S00026697). The restoration of extensively damaged teeth (for example as a consequence of traumatic injuries or caries) remains controversial despite decades of research. The amount of residual tooth structure and the defect location have been shown to have a significant role in the long-term prognosis of both tooth and restoration . SubgingSurgical crown lengthening is associated with esthetic and biologic disadvantages due to the lengthening of the clinical crown  and the While there is a wealth of data towards the survival and prognosis of tooth-supported fixed dental prostheses  or implaBesides efficacy , costs of a therapy are increasingly seen as a relevant aspect to consider during decision-making, as they are relevant for payers on all levels  but also providers. For orthodontic extrusion and restoration of extensively damaged teeth, any such health economic data is largely absent, and hence any comparison with the discussed alternatives (tooth extraction and replacement) is impossible when it comes to economic aspects.It was the aim of the present study to perform a health economic evaluation of orthodontically extruded and subsequently restored, extensively damaged teeth using routinely collected data of the discussed clinical study. The results of this study are of interest for patients, providers, and further decision-makers in dental healthcare.The clinical study was approved by the local ethics committee  and registered at the German Clinical Trials Register (DRKS registration number: DRK S00026697). Sample size calculation is described elsewhere . We hereStudy participants were consecutively recruited at the prosthodontic department at Charité–Universitätsmedizin in Berlin. The following inclusion criteria were defined: patients with (1) permanent teeth with subgingival or subcrestal defect, with (2) two proximal contact points, (3) pocket probing depths of ≤ 2 mm, (4) defects violating the biologic width and lacking the option for ferrule design preparation, (5) a prospective crown-root ratio not exceeding 1, (6) tooth mobility ≤ I, and (7) expected to require a single crown restoration. Patients of 18 years and older were assessed for eligibility. Both endodontically treated as well as vital teeth were included. Radiographs were obtained before treatment to assess the apical condition of the tooth and to determine the need for endodontic treatment or retreatment. Teeth with a remaining residual dentin thickness at the orifice of the canal of more than 1 mm and a ferrule height of 2 mm after extrusion were included. Exclusion criteria were (1) teeth with hypercementosis or (2) vertical root fractures, (3) molars, (4) tooth mobility ≥ II, (5) probing depths of ≥ 3 mm, and (6) teeth with prospective crown-root ratios exceeding 1. Patients should have been willing to appear for follow-up appointment over the next 5 years.n = 24). For one tooth , X-Post No3 (blue coding/1.67-mm diameter); for 15 teeth , X-Post No2 (red coding/1.47-mm diameter); and for 8 teeth , X-Post No1 (yellow coding/1.35-mm diameter) were used. Patients were contacted once a year during follow-up. Initial therapy procedures were performed under supervision of one operator (MB). Clinical and radiological re-examinations were performed by one operator (MB). Periodontal assessment was performed with the aid of a periodontal probe  under local anesthesia by the same operator. Radiographs were taken using paralleling technique. Analysis of radiographic images of extrusion and at recall is described elsewhere in detail [Figure n detail .Fig. 1InFor the present study, the following data were collected from electronic patient records: Employed restorative materials; initially performed restorative, endodontic, or periodontal therapies; possible restorative, endodontic, or periodontal re-treatments; and surgical or prosthetic retreatments including those leading to tooth loss and replacement. Furthermore, information on patients’ gender, age, and tooth type was collected.The health outcome was tooth retention in years, mainly as alternative outcomes  had not been measured, but also as the additional valuation of other health states  is only limitedly possible at present . SuccessThe study setting was German healthcare. A societal perspective was taken, reflecting on direct medical and non-medical costs as well as indirect costs (see below). In Germany, medical insurance is two-tiered, with most individuals (approx. 88%) being publicly insured (statutory insurance) and only a minority being privately insured. For members of the statutory insurance, nearly all dental procedures are fully covered, while only few need to be partially or fully paid out-of-pocket or by private  insurances. Forced orthodontic extrusion and the associated procedures are usually not covered by the public insurance, which is why direct medical costs were estimated using the private payer’s perspective.Using the outlined itemized treatments provided, costs were estimated on the basis of fee items of the German private insurance catalog GOZ (Gebührenordnung für Zahnärzte) . For GOZCosts were calculated in 2016 Euros, assuming all patients have been initially treated in 2016 (which is when the cohort was established). Costs during follow-up were discounted at 3% per annum, accounting for time preference  .p < 0.05. Statistical analysis and data visualization were performed with the aid of SPSS  and Excel 2016.Costs were calculated using a spreadsheet . Initial treatment costs and costs for follow-up procedures were calculated individually. Quantitative parameters were described using medians, minima, and maxima and 25% and 75% percentiles. Association of total initial treatment costs and cofounding variables was analyzed using generalized linear models. The significance level was set up A total of 30 patients  with a mean ± SD age of 52.5 ± 18.9 years were included, and a total of 35 teeth were restored. The mean observation time was 49.1 ± 18.9 months. Five patients (14%) dropped out during follow-up: Four patients were not available for follow-up appointments due to no valid contact data, and one patient due to death.In six patients and six teeth, complications arose. 1) Three teeth in three patients had to be extracted due to fractures: One upper first premolar fractured in a patient with a history of bruxism. Furthermore, two lateral upper incisors fractured. For both teeth, a narrow diameter of the root became evident after extrusion due to the anatomical taper of the extruded tooth. 2) In three patients, some degree of orthodontic relapse was documented. However, as restorations remained intact, they were not renewed. This relapse was clinically acceptable to both patients and the operator. Thus, survival rate was 91% and success rate 83%. Figure  In three Three teMean tooth survival was 46 ± 20 months. In three patients, costs during follow-up were particularly high as teeth were extracted and replaced. For two patients, teeth were replaced with fixed dental prostheses, and total costs were 1595 and 1694.9€, respectively . One patient received an implant-supported single crown accounting to overall costs of 4827.5€ .Initial direct medical costs did not differ between gender and age. Tooth position did not have a significant impact on direct medical costs. However, endodontic treatment as well as re-treatment and placement of post had a significant impact. Table Total costs were higher for initial treatment in comparison to follow-up. Figure This is the first study assessing treatment costs for retaining excessively damaged teeth using forced orthodontic extrusion and subsequent restoration. Overall, favorable survival rates and reasonable costs were observed for the majority of teeth. Two lateral incisors and one upper premolar had to be extracted due to fractures and were defined as failures. Notably, these teeth generated high costs for retreatment, mainly as replacement was expensive. Overall, costs were mainly generated initially. The findings of the present study need more in-depth discussion.First, according to the analysis, retaining excessively damaged teeth using forced orthodontic extrusion and subsequent restoration is likely to be less costly initially than tooth removal and subsequent implant placement in the chosen healthcare setting and perspective, where implant-supported crowns come with initial direct medical costs of around 4700€. Costs are comparable to a calculation using the national fee averages from the 2009 American Dental Association survey of dental fees . The finSecond, and along these lines, costs were associated with a range of aspects, most importantly the need for endodontic retreatment or post placement. Both, endodontic retreatment and post placement significantly influenced cost estimations. These findings are comprehensive as both treatment procedures are expenses that play a role in cost analysis in addition to the actual procedures of forced orthodontic extrusion. When informing patients towards treatment decisions, these aspects should be borne in mind. Patient’s age, gender, tooth type, and type of jaw did not influence treatment costs significantly. As gender and patient’s age did not affect tooth survival in long-term clinical trials investigating different post systems , these fThird, if complications arose, retreatment costs were considerable, mainly as tooth replacement was cost-intense. It is conceivable that failure risk increases over time, which is why even longer follow-up periods than the one in this study are needed to establish sound estimates of failure risk. Moreover, it may be that under a worst-case scenario, immediate tooth removal and replacement could be more cost-effective than the costly tooth retention via orthodontic extrusion. Comparative analyses are required to assess this relative cost-effectiveness ranking.This study has a number of strengths and limitations. First, it is the first study focusing on the economic evaluation of retaining teeth using orthodontic extrusion. Moreover, cost estimates reflected the true costs to payers and society in Germany. Second, and as a limitation, these costs may not apply to other settings and health care systems given the specific way they were derived. Third, our sample size was small, which may impact on the robustness of our cost estimates, but also the power to detect significant associations of various cofounding variables and costs. Fourth, treatment protocols were highly standardized, and all teeth were managed by the same dentists; clinical success but also costs may differ if other operators are considered . Moreover, the applied extrusion set described in the present study is currently no longer available for purchase. Therefore, a modified workflow with glass-fiber reinforced composite posts supplied by another manufacturer will be necessary in future research. Overall, and given the nature of this study, our findings should be interpreted with caution and require further confirmation.Within the limitations of this clinical study, total treatment costs for forced orthodontic extrusion and subsequent restoration of extensively damaged teeth were considerable. Costs were by large generated initially; endodontic and postendodontic therapies were main drivers. Costs for retreatments due to complications were limited as only few complications arose. Comparative cost-effectiveness evaluations are needed to establish economic rankings of different management strategies  for extensively damaged teeth.


Autoimmune diseases develop due to self-tolerance failure in recognizing self and non-self-antigens. Several factors play a role in inducing autoimmunity, including genetic and environmental elements. Several studies demonstrated the causative role of viruses; however, some studies showed the preventive effect of viruses in the development of autoimmunity. Neurological autoimmune diseases are classified based on the targets of autoantibodies, which target intracellular or extracellular antigens rather than neurons. Several theories have been hypothesized to explain the role of viruses in the pathogenesis of neuroinflammation and autoimmune diseases. This study reviewed the current data on the immunopathogenesis of viruses in autoimmunity of the nervous system. The immune system can identify and remove invading pathogens and prevent infection. Self-tolerance is described as a condition of the immune system that is not reactive to the self-antigen. A self-tolerance process is carried out throughout the immune system development, categorized as either central or peripheral tolerance . The breGiven the increasing evidence suggesting the association between viral infection and autoimmune disorders, and controversial data on the role of viruses in dysregulation of the immune response, herein, we aim to review the current data on the immunopathogenesis of the common viruses in developing nervous system autoimmune disorders.PubMed/Medline electronic database was searched using the keywords “Guillain–Barre syndrome”, “Myasthenia Gravis”, “autoimmune disease”, “Multiple sclerosis”, “experimental autoimmune encephalitis”, “central nervous system”, “COVID-19”, “HSV-1”, “Influenza”, “Epstein barre virus”, “EBV”, “CMV”, “Cytomegalovirus”, “Zika Virus”, and “varicella-zoster virus”. We reviewed the English articles with full-text available between January 1, 2000, to March 1, 2022. A total of 1210 articles were retrieved, and 283 articles were included in this study. The inclusion criteria for this narrative review were studies on the role of selected viruses in developing the neurological autoimmune diseases.The incidence of autoimmune diseases is estimated to be more than 5 percent in the general population, with an increasing prevalence in recent years . NeuroloAutoimmune diseases associated with the autoantibodies against extracellular epitopes, including cell surface and synaptic antigens, are less related to underlying malignancy. Moreover, in contrast to paraneoplastic CNS disorders, they are characterized by good responsiveness to immunosuppression. There is also a more complicated autoantibody expression pattern, mainly exposed during synaptic fusion and reuptake .Some theories have been postulated to explain the role of viruses in the pathogenesis of autoimmune diseases; however, the exact mechanism of viruses-induced neurological autoimmune disorders is still unknown. Altogether four main mechanisms were identified for viruses-induced neurological autoimmune disease: molecular mimicry, epitope spreading, bystander activation, and autoantibody production and immortalization of effector B-cells. Molecular mimicry is defined by similar antigens of self-epitopes and pathogen's antigens, resulting in a cross-reactive reaction of B and T cells to the self-antigen that caused autoimmune disease . The innIn this study, we sought to review the immunopathogenesis of viruses playing a role in the induction of autoimmunity of the nervous system, especially following the advent of the COVID-19 pandemic.Herpesviridae family, introduced in 1904. The virus infects 60 − 100% of people in adulthood [Cytomegalovirus (CMV), the linear double-stranded DNA virus, is a member of the dulthood , 15. Thedulthood . Moreovedulthood .Following viral penetration, pattern recognition receptors (PRRs) identify the pathogen-associated molecular pattern (PAMP) and the damage-associated molecular pattern (DAMP) of the virus, which induce the immune reaction to the virus . MoleculThere is also evidence suggesting the association between CMV and CD4 + CD28null T cells. Human studies have shown a direct relationship between the titer of CMV seropositivity and the number of CD4 + CD28 null T cells . In vitrFlaviviridae family, with a single-stranded positive-sense RNA genome, which can participate in the development of some central and peripheral nervous system disorders such as Guillain-Barré syndrome (GBS), transverse myelitis (TM), and meningoencephalitis [Zika virus is one of the members of the phalitis , 24. Altphalitis , few stuphalitis . It is sphalitis .Despite the effect of Zika virus in neurodevelopmental process and congenital pathologies are well understood , 28, theVaricella-zoster virus (VZV), known to cause chickenpox infection in humans, is one of the most frequent viral infections, affecting about 95% of adults in developed countries. After the initial infection, it establishes latency in the dorsal ganglia of most healthy people, which then might reactivate in particular circumstances . Some stMolecular mimicry, as an old hypothesis for explaining the possible role of viruses in inducing autoimmune diseases also has been suggested for the relationship between VZV and MS development. Degrees of molecular mimicry exist between VZV glycoprotein E and Heterogeneous Nuclear Ribonucleoprotein A1 (HNRNPA1), which is present in the nucleoplasm as it shares > 62% amino acid sequence similarity with the prion-like domain (PrLD) of HNRNPA1, signifying the reason behind autoantibodies against M9 and PrLD of HNRNPA1. HNRNPA1 mutation might stimulate the presence and enhancement of HNRNPA1 in the cytoplasm, along with the presentation of the protein by MHC-1, which all trigger a cascade of immune reactivation .Bearing in mind all considerations, the evidence favors the contributing role of VZV in inducing MS. However, further studies with more rigorous methodologies are needed to support this hypothesis..Epstein–Barr virus (EBV) is a ubiquitous member of the gamma-herpesvirus subfamily that is common in humans. The silent infection and the life-long persistence are the keys to the widespread infection of EBV in the human population. The virus is a linear DNA virus that encodes about 100 proteins and 44 micro RNAs (miRNAs). While many EBV miRNAs have no known function, there is evidence suggesting the role of viral miRNAs in innate immunity by regulating the inflammasome component NLRP3, the natural killer group 2D (NKG2D) ligand MICB, and the chemokine CXCL11 .Following initial infection, EBV crosses the Waldeyer’s ring to infect the naïve B cells. The activation of these naïve B cells to proliferating lymphoblasts is mediated via the Epstein-Barr nuclear antigen (EBNA). The activated lymphoblasts then migrate to germinal centers where they undergo a germinal center reaction to access their primary target (resting memory B cells) for latent persistence. The signals from the EBV encoded latent membrane proteins (LMPs) contribute to the survival of these infected lymphoblasts. It is noteworthy that given the type of latency, the virus expresses different sets of latent products including LMP1 and LMP2 , 40.Sumaya et al. were the first ones who described an increased frequency of antibodies to EBV in patients with MS compared to healthy controls. Since then, many studies have demonstrated increased antibodies against EBV antigens titer in MS patients [Bjornevik and Cortese et al. utilized longitudinal evaluation of over 10 million adults between 1993 and 2013 to demonstrate the association between EBV infection and MS development. They showed a 32-fold increase in the risk of MS following EBV infection, but the risk was not increased after other viral infections. Moreover, neurofilament light chain levels were increased only after EBV seroconversion.It is believed that EBV-associated pathologies result from the disruption of the virus-host immune system balance, and clinical manifestations of EBV infection emerge as a result of provoked immune response rather than EBV itself. In this regard, several studies have shown the immune response to EBV is disturbed in MS patients. patients –49. Morepatients . InteresNotably, many hypotheses have been proposed to express the role of EBV in developing MS. Molecular mimicry has repeatedly been suggested as a potential pathogenic mechanism. The LMP1 mimics CD40 receptors, which play a role in B and T-cells interactions, and LMP2A mimics B-cell receptors. Moreover, an IL-10-like cytokine that EBV produces is crucial to B-cell activations . In addiFurthermore, it seems there is a relationship between EBV-infected B cells and the development of MS. Notably, EBV micro RNAs can protect EBV-infected B cells from CD8 + T cell response by reducing the EBV-specific CD8 + T cell proliferation and IFN-γ secretion . InteresPender et al. applied the vitro-expanded autologous EBV-specific CD8 ( +) T cells directed against viral latent proteins to treat a patient with secondary progressive MS. Their results were promising with no adverse effects and evidence of clinical and MRI improvement. Since then, many efforts have been made in this regard. More ever, there is a possibility that currently available B cell depleting therapies might be regarded as anti-EBV therapies, which deplete circulating memory B cells, the primary site of latent EBV infection [Interestingly, regarding the role of EBV in the development of MS, particular attention has been paid to treatment strategies in this field. For the first time in 2014, nfection . The mecRetroviridae family, the Orthoretrovirinae subfamily, and the deltaretrovirus genus, preferentiallyinfects CD4+ T cells in vivo [The human T-lymphotropic virus type 1 (HTLV1) is a single-stranded RNA virus that belongs to the  in vivo . The HTLHTLV1 affects approximately 5–10 million persons worldwide. Most infected individuals remain asymptomatic; however, a portion of HTLV-1-positive individuals will develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), adult T-cell leukemia/lymphoma (ATLL) disease, and HTLV-1-Associated Uveitis . HAM / T+CCR4+ T cells that coexpressed the Th1 marker CXCR3 and produced T-bet and IFN-γ were present in the CSF and spinal cord lesions of HAM/TSP patients [In HAM/TSP patients, the Tax mRNA level is significantly higher than in healthy people . The Taxpatients . Being apatients .herpes simplex family with a double-stranded DNA-encapsulated virus [Herpes simplex virus type 1 (HSV-1) is a part of the ed virus . HSV-1 ied virus . The HSVed virus . During ed virus . On the ed virus .The mechanisms leading to latency and reactivation and which are the viral and host factors controlling these processes are not completely understood. The Us6 gene encodes glycoprotein D, which is a part of HSV construction is required for penetrating the host cells, and inhibiting the apoptosis by activating NF-κB and enhancing NF-κB-dependent anti-apoptotic genes such as FLIP and c-IAP2 , 86. GlyIn terms of neurotoxicity, herpes simplex encephalitis (HSE) is considered as the most devastating manifestation of HSV-1. It can occur either in primary infection or upon reactivation from latency. Cytolytic HSV-1 proliferation and immune factors are involved in the development of HSE –97. The Interestingly, there is evidence suggesting the potential link between HSV and Alzheimer's disease (AD). HSV-1 DNA has been shown to be co-located with amyloid B in the brain tissue samples of patients with AD. The association between HSV-1 and AD is stronger in individuals carrying the APOE4 allele, one of the strongest genetic risk factors for AD . MoreoveRegarding the association of HSV1 and MS, the scope of article is inflammatory disorders.orthomyxoviridae family membership [Influenza virus is an enveloped, negative-sense single-stranded RNA virus that consists of 8 parts and is classified as the mbership . Influenmbership . Currentmbership . HA bindmbership . Infectimbership . Howevermbership . Influenmbership . A case–mbership . Howevermbership . Among tmbership . Sivadanmbership . In anotmbership .The influenza virus genome is detected by TLR7, while during virus replication, double-stranded RNA is detected by TLR3. Activation of the corresponding TLRs by ssRNA or dsRNA activates the signal cascade. TLRs are not required to activate T cells against influenza. However, they induce B cell responses directly and indirectly by INF-α , which scoronaviridae family [SARS-CoV2 is an enveloped, positive-sense, single-stranded RNA (ssRNA) of the e family –130. Core family , 132 ande family , 134. Ste family , AD [136e family . So far,e family . It was e family . The fire family . Recentle family , 140. Poe family , 141. Ine family . Howevere family . In one e family , 145. Ine family . Li S ane family . In a ree family . Relatede family . It was e family , 103.SARS-CoV-2 requires the angiotensin-converting enzyme 2 (ACE2) as a functional receptor to penetrate cells. The virus binds to the ACE2 receptor via the spike, although the virus has also been observed to use Basigin (CD147) and Neuropilin-1 (NRP1) as receptors . After SMolecular mimicry is the one of the main proposed mechanism involved in neurodegeneration which can cause the immune system to become overactive in autoimmune diseases are similar to those of the immune response against SARS-CoV2 . MoleculFollowing the emergence of covid-19, vaccination against SARS-Cov-2 is considered as an effective strategy for preventing infected to covid-19 and decreasing the severity and mortality of patients infected by SARS-Cov-2. However, some clinical studies reported the patients with novel neurological autoimmune diseases who is diagnosed after receiving covid-19 vaccines. Rinaldi et al. reported the patients with CNS inflammatory demyelinating events following covid-19 vaccine administration. They showed that covid-19 vaccine can induce acute transverse myelitis, MS, acute demyelinating encephalomyelitis (ADEM), and neuromyelitis optica spectrum disorder (NMOSD) . MoreoveAutoimmune diseases are frequently developed based on the interaction between several factors, including genetic susceptibility, aberrant immune response, and environmental factors such as infections. The viral infection seems more critical to inducing autoimmune disease disorders. The exact level of involvement of these factors was not elucidated ; however


The mosquito must survive long enough after ingesting the pathogen to deliver infectious bites. Significant levels of pathogen-associated mortality in this host would have a detrimental impact on transmission. Here, we show that Aedes aegypti with a genetic lesion in Dicer-2 are acutely susceptible to multiple viruses associated with human disease, but not other types of infections. Further, we demonstrate that the pathology associated with these infections is primarily controlled through an RNA interference pathway that functions as a resistance mechanism. These results reveal insights into the transmission of many important diseases.Models of mosquito-borne disease transmission illustrate that the longevity of the insect vector is a particularly powerful factor in the calculation of basic reproduction number (R Aedes aegyptiDicer-2 (Dcr-2) gene renders the insect acutely susceptible to a disease phenotype upon infection with pathogens in multiple virus families associated with important human diseases. Additional interrogation of the disease phenotype demonstrated that the virus-induced pathology is controlled through a canonical RNA interference (RNAi) pathway, which functions as a resistance mechanism. These results suggest comparatively modest contributions of proposed tolerance mechanisms to the fitness of A. aegypti infected with these pathogens. Similarly, the production of virus-derived piwi-interacting RNAs (vpiRNAs) was not sufficient to prevent the pathology associated with viral infections in Dcr-2 null mutants, also suggesting a less critical, or potentially secondary, role for vpiRNAs in antiviral immunity. These findings have important implications for understanding the ecological and evolutionary interactions occurring between A. aegypti and the pathogens they transmit to human and animal hosts.While it has long been known that the transmission of mosquito-borne viruses depends on the establishment of persistent and nonlethal infections in the invertebrate host, specific roles for the insects’ antiviral immune pathways in modulating the pathogenesis of viral infections is the subject of speculation and debate. Here, we show that a loss-of-function mutation in the  While t0) , 3. At t0) . HoweverDrosophila melanogaster with genetic lesions in Dicer-2 (Dcr-2) were essential to demonstrating the nuclease’s role in the production of virus-derived siRNAs and the initiation of a protective antiviral immune response , a pathogen-associated molecular pattern commonly associated with viral infection –7. Drosoresponse , 8, 9. Eresponse , 8, 10. response . One strresponse , 13. An response . The gui. A characteristic enrichment of adenine residues at the 10th position, or so called A10 nucleotide (nt) bias, in ping-pong dependent piRNAs has long been the hallmark of products derived by this mechanism in the Drosophila germline  strands, which may be detected in large sequencing datasets through bioinformatic approaches (There is evidence to suggest that a ping-pong-dependent piwi-interacting RNA (piRNA) pathway, which functions independently of Dcr-2, may also target RNA viruses in the soma of disease vector mosquito species –23. A chgermline . Anothergermline . As cleagermline . In Drosproaches –27.A. aegypti include Zika virus, dengue virus serotypes 1 to 4 (DENV 1-4), Japanese encephalitis virus, yellow fever virus (YFV), and chikungunya virus (CHIKV), which are collectively responsible for hundreds of millions of cases of human morbidity each year. Although the transmission of these viruses depends on the establishment of a persistent, nonlethal infection in the insect vector host, the mechanism or mechanisms by which pathogenesis is modulated in A. aegypti remains a topic of speculation and debate. Inhibiting the production of siRNAs with the well-known flock house virus suppressor of RNA silencing, B2, was shown to increase mortality in mosquitoes infected with a recombinant Sindbis virus (SINV), but these experiments could not entirely rule out the possibility that the observed phenotype in mosquitoes might be due to some hitherto undescribed function of the B2 protein  contributing to the resistance of D. melanogaster to various fruit fly pathogens  was not sufficient to prevent a disease phenotype in the arbovirus-infected Dcr-2 null mosquitoes, also suggesting a less critical, or potentially secondary, role for vpiRNAs in antiviral immunity. These findings have important implications for understanding the transmission of pathogens that impose a significant burden on global public health.Here, we interrogated the role of athogens , 31–35. Dcr-2 null mutant A. aegypti by gene editing has been described (A. aegypti “sensor” strain has been described (Dcr-2 mutants were screened for expression of eye-specific enhanced green fluorescent protein (EGFP) 6 d after emergence of adults. All mosquito infections were performed by injection of 0.5 µL (106 pfu/mL) YFV (ASIBI), DENV-2, DENV-4, or SINV prepared in Dulbecco’s modification of Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum, nonessential amino acids, L-glutamine, and antibiotics into the thoraces of adult mosquitoes. Mock-infected mosquitoes were inoculated with the same volume of DMEM. Liverpool strain or wild-type sibling mosquitoes (scored as negative for EGFP expression) served as controls. Three-to-five-day-old A. aegypti were fed on a blood meal containing 6.7 × 106 pfu/mL SINV. Blood meals were prepared by mixing equal volumes of defibrinated sheep erythrocytes and virus suspension. Blood meals were heated to 37 °C and administered to mosquitoes with a Hemotek feeding apparatus. Mosquitoes were permitted to feed for 1 h and then fully engorged females were separated and held in individual chambers. A portion of each infectious blood meal was frozen and back titers determined by plaque assay. Liverpool, Dcr-2 null mutant, or wild-type sibling A. aegypti strains were incubated at 28 °C and 80% relative humidity, with a 14/10-h day/night light cycle. Assays for infectivity were performed as described previously (E. coli (DH5a) and Staphylococcus aureus (S. aureus), representing gram-negative and gram-positive bacteria, respectively, were used as previously described  of adult female mosquitoes 96 h after infection with Tri Reagent RT (Molecular Research Center) according to the manufacturer’s instructions. The levels of positive sense viral RNAs present in the samples were determined by strand-specific quantitative real-time PCR (RT-qPCR) using Taqman assays (Life Technologies) and a standard curve, as described previously . The spehttp://hannonlab.cshl.edu/fastx_toolkit/) and identifiable noncoding RNA sequences  removed with Bowtie (https://bioconductor.org/packages/release/bioc/html/edgeR.html) was used to determine differential expression between replicate datasets. Raw sequence counts with less than ten copies per million reads were excluded from the input, with all identifiable reads  being used to normalize the datasets through the TMM method  according to the manufacturer's instructions. All independent biological replicates in a cohort were multiplexed and sequenced on a single lane of a HiSeq (Illumina) flow cell by the Next Generation Sequencing and Microarray Core Facility, The Scripps Research Institute, La Jolla, California. Bioinformatic analysis of libraries was performed as described previously . Brieflyh Bowtie . RemainiM method . NucleotM method . As the t test, minimum three independent biological replicates and three technical machine replicates. Infection data were analyzed with a two-tailed unpaired t test, minimum three independent biological replicates. Survival curves were generated by the Kaplan–Meier method, with data for either two or three groups being compared by the log-rank (Mantel–Cox) test. Differential expression between normalized replicate small RNA datasets was analyzed with the edgeR software package as described above. Z-scores for the number of small RNA pairs found with overlaps ranging from 1-20 nucleotides were calculated with a previously published formula  gene was edited with a pair transcription activator-like effector nucleases generating a 17 base pair deletion in exon 5 (A. aegypti sensor strain (kmo mutant (white eye) strain is transformed with a construct containing three independent, but identical, eye-specific 3xP3 promoters. The first promoter expresses the red fluorescent protein derived from Discosoma sp. (DsRed), which serves as a marker of transformation. The second 3xP3 promoter expresses EGFP. While the expression of EGFP from this promoter would normally result in an eye-specific EGFP+ phenotype in this white-eyed strain, the third 3xP3 promoter in the series expresses an inverted repeat sequence with homology to a portion of the EGFP mRNA. Thus, the eye-specific EGFP+ phenotype is only observed upon inactivation or impairment of RNA silencing, which prevents the siRNA pathway from processing the dsRNA substrate formed by the inverted repeat sequence and silencing EGFP . Thus, eye-specific expression of EGFP also proved to be useful as a phenotypic marker for screening and selecting adult mosquitoes homozygous for the mutant Dcr-2 allele. The survival of homozygous null mutants, identified through eye-specific EGFP expression levels, over a 2-wk time period was not significantly different from that of wild-type siblings or Liverpool strain A. aegypti, indicating that the loss of function mutation had little or no adverse effect on the vigor of the mutants in the absence of microbial challenge . Further, challenge of Dcr-2 null mutant A. aegypti with both gram-negative and gram-positive bacteria did not reveal a general susceptibility to nonviral pathogens . However, consistent with results reported in a previous study with D. melanogaster . Thus, additional studies may be merited in order to investigate possible interactions between Dcr-2 and the Toll pathway in A. aegypti.The n exon 5 A. Mosquin exon 5 B, were sr strain . Brieflying EGFP . Strong 2 allele C, indicanogaster , Dcr-2 nDcr-2 null and wild-type sibling A. aegypti with SINV. In comparison to wild-type siblings, Dcr-2 null mutants exhibit a clear disease phenotype (Movie S1), characterized by significantly elevated levels of virus replication .We next sequenced and analyzed small RNAs present in ll lines –22, two ll lines C. The fill lines C and D. ll lines C. These Dcr-2 null mutant A. albopictus cell lines suggested that an antiviral response directed solely by vpiRNAs might be capable of modulating alphavirus pathogenesis in the absence of siRNA production , this increase was not sufficient to prevent mortality, at least not to the same extent observed in wild-type or heterozygous siblings infected with equivalent titers of virus . While these products cannot be confirmed as piRNAs, the extremely limited number of reads may have prevented any meaningful analysis of overlapping pairs, and also may have prevented the identification of specific biases as well.Previous work in oduction . While with SINV E, which of virus B. Howeveof virus F. These th CHIKV . HoweverDcr-2 null and wild-type sibling mosquitoes in an infectious blood meal. Loss of functional Dcr-2 did not increase the prevalence of SINV infections in comparison with wild-type siblings 6 d after virus ingestion , but did result in increased dissemination of the virus at 8 d post infection  was statistically indistinguishable from that of wild-type siblings ingesting an equivalent titer of SINV . This result indicates that pathogenesis requires escape from the midgut and the establishment of a systemic infection. Thus, the siRNA pathway appears to be essential both for limiting dissemination of the virus from the midgut tissue, and modulating the pathogenesis of the systemic mosquito infections necessary for disease transmission. Interestingly, the pathogen load in Dcr-2 null mutants that succumbed to systemic infections 6 to 10 d after ingesting the virus did not significantly differ from those of mutants succumbing 11 to 21 d after virus ingestion . Thus, the observed differences in the longevity of the systemically infected mutants suffering under similar levels of viral burden may represent evidence of genetic variation in tolerance mechanisms, and in the future, this would be interesting to investigate further.In order to confirm that the disease phenotype observed previously was not somehow specific to the route of infection, we next administered SINV to ngestion A. Howeveme point B. This snfection . Importasquitoes C and D. Dcr-2 null background . While the biogenic products of the antiviral siRNA pathway are clearly visible in wild-type siblings infected with YFV, evidence of processing by the piRNA pathway is much less obvious . Thus, in the absence of a functional antiviral RNAi pathway, genetic variation in tolerance traits might explain the observed differences in longevity. However, according to the theory currently supported in plants, resistance and tolerance should be under correlational selection as they are mutually redundant traits Click here for additional data file.Movie S1.Ae. aegypti.Pathogenic alphavirus infection in RNAi deficient  Side by side time course comparison of Dcr-2 null mutant and wild-type sibling mosquitoes after infection with μ500 pfu of Sindbis virus.


Canis lupus familiaris). Previous studies have documented hybridization between wild canids and domestic dogs in North America and Europe. However, there have been no reports of such hybridization in South America until now. In 2021, a female canid from Vacaria in Rio Grande do Sul State, Brazil, was brought to the Center for Conservation and Rehabilitation of Wild Animals—Preserves. This animal exhibited unusual phenotypic characteristics, displaying intermediate traits between domestic dogs and wild canids. Based on these observations, we hypothesized that this animal might result from interspecific hybridization. Therefore, this study aimed to test this hypothesis using genetic and cytogenetic approaches. Our analysis suggests that the canid under investigation is a hybrid between the pampas fox and domestic dog, but future studies are necessary to investigate additional cases of this hybridization in nature. Therefore, the combination of genetic and cytogenetic markers proved valuable in elucidating this case of hybridization. To our knowledge, this represents the first documented case of hybridization between these two species.In South America, the natural habitats of wild canids have undergone significant environmental disruptions, leading to interactions between these animals and domestic dogs (Canis familiaris) and that of the pampas fox , was found. Our analysis suggests that the animal is a hybrid between a domestic dog and a pampas fox, but future studies are necessary to investigate additional cases of this hybridization in nature. This finding worries for the conservation of wild canids in South America, especially concerning Lycalopex species. Hybridization with the domestic dog may have harmful effects on pampas fox populations due to the potential for introgression and disease transmission by the domestic dog. Therefore, future studies to explore the consequences of hybridization on genetics, ecology, and behavior of wild populations will be essential to improve the conservation of this species.Hybridization between species with different evolutionary trajectories can be a powerful threat to wildlife conservation. Anthropogenic activities, such as agriculture and livestock, have led to the degradation and loss of natural habitats for wildlife. Consequently, the incidence of interspecific hybridization between wild and domestic species has increased, although cases involving species of different genera are rare. In Vacaria, a Southern city in Brazil, a female canid with a strange phenotype, which had characteristics between the phenotype of the domestic dog ( Interspecific hybridization among animals has increased over the years, such as in mammals , birds , and fisAtelocynus, Cerdocyon, Chrysocyon, Speothos, Urocyon, and Lycalopex . Ne. NeLycalanid see .L. gymnocercus. However, upon reviewing these photographs, we did not come across any specimens with a pelage color resembling that of the canid found in Vacaria City.We discovered a total of 1112 photographs on INaturalist that were registered as Canis lupus familiaris) and a pampas fox . This discovery implies that, although these species diverged about 6.7 million years ago [This study investigated an intriguing canid individual exhibiting unusual phenotypic characteristics, which could not be associated with any known canid species with distribution in Brazil, according to our photographs analysis. Using genetic and cytogenetic markers, our findings suggest that this individual represents a first-generation hybrid between a dog  and C. thous (2n = 74) would produce a hybrid with 76 chromosomes, but most of the autosomal chromosomes in C. thous are bi-armed, which is not characteristic of the Canid karyotype, effectively discarding this possibility.The analysis of mtDNA revealed that the maternal lineage of the canid belonged to the pampas fox. Additionally, five APOB, FES, and GHR segment polymorphisms were consistent with our cytogenetic and mtDNA data. These segments are specific to each species, so the parental species (dog and pampas fox) exhibited homozygote sites, while the canid showed a heterozygote site with both dog and pampas fox polymorphisms. Although the observed indel in the FES segment is also found in two canids,  species . MoreoveAlthough our nuclear analysis was limited to only two specimens of the putative parental species, it does not undermine our evidence of hybridization. While it does not offer sufficient information to definitively determine whether the observed genotypes in the canid are the result of hybridization or intraspecific variation within the pampas fox population, it does provide valuable insights into the hybridization event. When we consider our integrative analysis, which includes photographs, cytogenetic data, mtDNA analysis, and nuclear analysis, the possibility of hybridization between the pampas fox and the dog should not be disregarded.The Pampas biome represents a large proportion of the pampas fox distribution range. However, this species is also common in open woodlands and in modified habitats, such as grazed pastures and croplands . The geoCanis lupus familiaris. Further studies are necessary to examine the frequency of hybridization and the potential for genetic introgression of dog genes into Pampas fox populations once the occurrence of introgression of dog alleles to pampas fox populations means introgression of alleles shaped by artificial selection on species vulnerable to natural selection. The pampas fox has a coat color very similar to its habitat, as shown in This study suggests a unique case of hybridization between the pampas fox and the domestic species In conclusion, here we suggest the first report of hybridization between a domestic dog and a pampas fox. Genetic management of interspecific hybrids is important to species conservation. This step requires the application of methodologies able to provide an easy and undoubted genetic characterization of parents and hybrids . In this


Here, we demonstrate that the 3CLprores virus replicates efficiently in the lungs of intranasally infected female Syrian hamsters and causes lung pathology comparable to that caused by the WT virus. Moreover, hamsters infected with 3CLprores virus transmit the virus efficiently to co-housed non-infected contact hamsters. Importantly, at a dose of 200 mg/kg (BID) of nirmatrelvir, the compound was still able to reduce the lung infectious virus titers of 3CLprores-infected hamsters by 1.4 log10 with a modest improvement in the lung histopathology as compared to the vehicle control. Fortunately, resistance to Nirmatrelvir does not readily develop in clinical setting. Yet, as we demonstrate, in case drug-resistant viruses emerge, they may spread easily which may thus impact therapeutic options. Therefore, the use of 3CLpro inhibitors in combination with other drugs may be considered, especially in immunodeficient patients, to avoid the development of drug-resistant viruses.The SARS-CoV-2 main protease (3CLpro) is one of the promising therapeutic targets for the treatment of COVID-19. Nirmatrelvir is the first 3CLpro inhibitor authorized for treatment of COVID-19 patients at high risk of hospitalization. We recently reported on the in vitro selection of SARS-CoV-2 3CLpro resistant virus (L50F-E166A-L167F; 3CLpro Characteristics of drug-resistant SARS-CoV-2 viruses are unclear. Here, the authors report that an in vitro selected nirmatrelvir-resistant virus replicates efficiently and can be transmitted in Syrian hamsters but that nirmatrelvir retains some antiviral activity against that virus in infected hamsters. So far, three antiviral drugs have been approved/authorized by FDA and EMA for clinical use in COVID-19 patients i.e. the nucleoside analogs remdesivir and molnupiravir and the viral protease (3CLpro/Mpro) inhibitor nirmatrelvir3. Interestingly, the cleavage site of the SARS-CoV-2 3CLpro substrate is not recognized by any known human proteases5. Therefore, 3CLpro is a selective antiviral drug target. Nirmatrelvir (PF-07321332) is a peptidomimetic reversible covalent inhibitor of 3CLpro that is co-formulated with the pharmacokinetic enhancer ritonavir (the resulting combination being marketed as Paxlovid)6. When treatment is initiated during the first days after symptom onset, it results in roughly 90% protection against severe COVID-19 and hospitalization7. Besides nirmatrelvir, the non-peptidic, non-covalent inhibitor, ensitrelvir  has been approved in Japan for emergency use against SARS-CoV-28. Other 3CLpro inhibitors are also currently in clinical development.SARS-CoV-2 3CLpro is a cysteine protease which cleaves the two viral polyproteins (pp1a and pp1ab) at eleven different sites, resulting in various non-structural proteins, which are essential for viral replication12. Moreover, transmission of drug resistant viruses has also been reported for HIV13 and influenza virus14. Selection of remdesivir-resistant SARS-CoV-2 variants has been also reported in cell culture and in clinical settings17. On the other hand, there are no clear data available yet about emergence of resistant virus variants to molnupiravir or nirmatrelvir in treated patients. Recently, we reported on in vitro selection of a SARS-CoV-2 resistant virus against a first generation 3CLpro inhibitor (ALG-097161) that is cross-resistant to several 3CLpro inhibitors including nirmatrelvir18. The identified resistant virus carries three amino acid substitutions in 3CLpro (L50F-E166A-L167F) that result in a more than 20 fold increase in EC50 values for different 3CLpro-inhibitors18. These substitutions are associated with a significant loss of the 3CLpro activity in enzymatic assays, suggesting a subsequent reduction in viral fitness18.The emergence of drug-resistant viruses is a major concern when using antivirals. Development of drug resistant variants has been reported during antiviral treatment against different infections including with the human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpesviruses and influenza virusres) virus in Syrian hamsters, (ii) assess the transmission potential of this in vitro selected drug resistant virus from intranasally infected index hamsters to non-infected contact hamsters and (iii) evaluate the efficacy of nirmatrelvir against this resistant variant in the hamster infection model.Here, we aimed to (i) explore the infectivity and virulence of the in vitro selected 3CLpro (L50F-E166A-L167F) nirmatrelvir resistant (3CLprores) virus in animals, two groups of index hamsters (each n = 12) were intranasally infected with 1 × 104 TCID50 of either the wild-type (WT) SARS-CoV-2 virus (USA-WA1/2020) or the reverse engineered nirmatrelvir-resistant virus carrying substitutions L50F-E166A-L167F in 3CLpro18. On day 1 post-infection (pi), each of the index hamsters was co-housed in a cage with a contact hamster. The co-housing continued until 3 days after start of contact  nirmatrelvir-resistant  and (2.2 × 103 − 1.1 × 107) genome copies/mg lung tissue, respectively  twice daily by oral gavage for 4 consecutive days, starting just before the infection [with the WT or the 3CLprores virus (Day 0)]. On day 4 pi, all animals were euthanized and lungs were collected to quantify the infectious virus titers and evaluate the lung pathology  resulted in a reduction of viral replication (1.4 log10) as compared to the corresponding vehicle group  of lung histopathology scores (close the baseline score of uninfected untreated hamsters) was observed in the nirmatrelvir -treated group infected with the WT virus  resulted in slight (non-significant) reduction of lung histopathology scores , as compared to the other groups (mean % body weight change <2)  Fig. . Sequenc<2) Fig. . Passagi19. We have recently reported on the in vitro selection of resistant SARS-CoV-2 against a number of 3CLpro inhibitors including nirmatrelvir and ensitrelvir. This virus carries the L50F-E166A-L167F substitutions in the 3CLpro protein18. In a FRET-based assay, the enzymatic activities of recombinant 3CLpro proteins carrying the L50F, E166A and L167F substitutions alone or combined proved to be significantly lower, compared to that of the WT protein18. In another similar study, in vitro selection of resistant variants against nirmatrelvir resulted in identification of several resistance-associated substitutions with the E166V substitution resulting in the strongest resistance phenotype20. However, this substitution at position 166 resulted in loss of the virus replication fitness in vitro that was restored by compensatory substitutions such as L50F and T21I20.So far SARS-CoV-2 drug resistant variants have only been reported in patients treated with the viral polymerase inhibitor remdesivirres virus resulted in efficient replication of the virus in the lungs. However, the viral RNA loads and infectious titers in the lungs of hamsters infected with the 3CLprores virus were 0.6 and 1.9 log10 lower compared to the WT virus-infected index hamsters. The relative lower infectious virus titers as compared to the RNA levels of the 3CLprores virus (isolated from the lungs of hamsters) seems to indicate that this 3CLprores virus has a lower relative infectivity  than the WT virus. We have as yet no explanation of this observation. On the other hand, both the WT and the 3CLprores virus caused a comparable lung pathology in the infected index hamsters.Here, we assessed the in vivo fitness of this 3CLpro (L50F-E166A-L167F) nirmatrelvir (and other protease inhibitors) resistant virus in terms of infectivity and transmission potential in a hamster infection model. Intranasal infection of female Syrian hamsters with either the WT or the 3CLprores virus, in a cage with a contact (non-infected hamster) for 3 days revealed efficient transmission of either virus between infected and non-infected hamsters. All contact hamsters from both groups had detectable viral RNA loads in their lungs. Infectious virus titers were detected in the lungs of respectively 92% and 75% of the sentinels co-housed with animals that had been infected with either the WT virus or 3CLprores virus. The discrepancy between the number of contact hamsters with viral RNA in the lungs and detectable infectious virus in their lungs might be explained by the fact that all the contact animals were sacrificed at the same day (day 3 post first contact) whereas transmission most likely does not occur in a synchronized way; some animals may possibly soon, others later after contact be infected. Accordingly, for animals that acquired the virus rather late after the first contact, viral RNA might already be detectable in the lungs, but one or two more days may have been required to allow detection of infectious titers.Co-housing of each index animal, infected with either the WT or the 3CLprores virus versus the WT virus in the hamster model. Although we observed a marked reduction in the antiviral efficacy of nirmatrelvir against the resistant virus (compared to WT), the compound was still able to reduce the infectious virus titers to some extent (by 1.4 log10) at 200 mg/kg dose. A similar pattern of reduced nirmatrelvir efficacy against the drug-resistant virus was also observed in the lung histopathology scores. In an earlier study in which we studied the effect of nirmatrelvir in the SARS-CoV2 hamster infection model, a slightly higher dose of the compound was used (i.e. 250 mg/kg instead of 200 mg/kg in the present study)21. This 250 mg/kg dose resulted in peak plasma levels of 22.4 ± 11 µM21 and peak lung exposure of 6.2 ± 4.9 µM . In Vero E6, the EC50 of nirmatrelvir against the 3CLprores virus is 6.1 µM18. With the 200 mg/kg dose (used in the current study), lung exposure may be expected to be somewhat below 6 µM; which may explain the residual activity of this dose against the resistant virus. Modeling based on human pharmacokinetic data of nirmatrelvir will be needed to estimate whether nirmatrelvir treatment may still retain some efficacy in patients carrying such a drug-resistant virus.We also explored the antiviral efficacy of nirmatrelvir against the 3CLpro14 although it should be mentioned that the barrier to resistance of these amantadines is much lower than that of the SARS-CoV-2 3CLpro inhibitors currently being developed. For the treatment of infections with HIV and HCV fixed-dose combinations prevent the emergence of resistant variants22. It will be prudent and important to explore the efficacy and impact on potential resistance development of combinations of SARS-CoV-2 3CLpro inhibitors with drugs with a non-overlapping resistance profile.Taken together, these results show that the 3CLpro (L50F-E166A-L167F) nirmatrelvir resistant virus is able to efficiently replicate in the lungs of Syrian hamsters and that this virus can be transmitted via direct contact to co-housed naive hamsters. Nirmatrelvir is less efficient in animals infected with the resistant virus than in WT-infected hamsters, but still retains a certain level of antiviral activity. Two of the selected substitutions (i.e. L50F and L167F) have been reported to be detected (at low frequencies) in SARS-CoV-2 viruses naturally circulating in the population (17). Moreover, the Paxlovid label indicates that the E166V subsititution is among the treatment-emergent substitution, which is more common in nirmatrelvir/ritonavir treated subjects relative to placebo-treated subjects. These observations suggest that the in vitro selected 3CLpro (L50F-E166A-L167F) resistant virus may also emerge in treated patients with increased use of Paxlovid or other 3CLpro targeting drugs in the future. The emergence of drug-resistant SARS-CoV-2 variants that can be efficiently transmitted in the population is of serious concern. It may result in reduced potency of an important therapeutic option for patients that acquire the drug-resistant virus. Efficient transmission of the anti-influenza drugs amantadine and rimantadine has been well documentedres) virus (passage 1 on Vero E6 cells) used here has been described before18. Live virus-related work was conducted in the high-containment A3 and BSL3 facilities of the KU Leuven Rega Institute (3CAPS) under licenses AMV 30112018 SBB 219 2018 0892 and AMV 23102017 SBB 219 20170589 according to institutional guidelines. Only authorized personnel that have received intensive training have strictly controlled access to these facilities.The WT SARS-CoV-2 strain used in this study, SARS-CoV-2 USA-WA1/2020 , was obtained through BEI Resources (ATCC) cat. No. NR-52281, batch 70036318. This isolate has a close relation with the prototypic Wuhan-Hu-1 2019-nCoV (GenBank accession 112 number MN908947.3) strain as confirmed by phylogenetic analysis. The reverse engineered 3CLpro (L50F-E166A-L167F) nirmatrelvir resistant  were cultured in minimal essential medium  supplemented with 10% fetal bovine serum (Integro), 1% non-essential amino acids , 1% L- glutamine (Gibco) and 1% bicarbonate (Gibco). End-point titrations on Vero E6 cells were performed with medium containing 2% fetal bovine serum instead of 10%. Cells were kept in a humidified 5% CONirmatrelvir was synthesized at Excenen (China). In addition, a 1:1 MTBE solvate form of nirmatrelvir was kindly provided by Pfizer. The Excenen batch was used for the first experiment and the MTBE solvate form Pfizer was used for the second experiment. Nirmatrelvir (Excenen) was formulated as 100 mg/ml in a vehicle containing 43% ethanol, 27% propyele glycol (Sigma) in sterile distilled water. The MTBE solvate form (Pfizer) was formulated as 40 mg/ml suspension in 2% (v/v) Tween80 in 98% (v/v) of 0.5% (w/v) Methyl Cellulose by geometric dilution.24. Female Syrian hamsters (Mesocricetus auratus) were purchased from Janvier Laboratories and kept per two in individually ventilated isolator cages  at 21 °C, 55% humidity and 12:12 day/night cycles. Housing conditions and experimental procedures were approved by the ethics committee of animal experimentation of KU Leuven (license P065-2020). For infection, two groups of index female hamsters of 6-8 weeks old were anesthetized with ketamine/xylazine/atropine and inoculated intranasally with 50 µL containing 1×104 TCID50 of either SARS-CoV-2 WT virus or the 3CLprores virus(day 0). In the morning of day 1 post-infection (pi), each index hamster was co-housed with a contact (sentinel) hamster (non-infected hamsters) in one cage and the co-housing continued until the sacrifice day i.e. 3 days after start of exposure. On day 4 pi, animals were euthanized by intraperitoneal injection of 500 μL Dolethal  and lungs were collected for further analysis. Two independent studies were performed each with a total of n = 12 per group.The hamster infection model of SARS-CoV-2 has been described beforen = 12 for vehicle groups and n = 11 for 200 mg/kg nirmatrelvir groups. For 100 mg/kg dose group, a single experiment was performed with n = 6.Female Syrian hamsters were treated by oral gavage with either the vehicle or nirmatrelvir at 100 or 200 mg/kg/dose twice daily (BID) starting from D0, just before the infection with either SARS-CoV-2 WT virus or the 3CLprores virus (day 0) as described above. All the treatments continued until day 3 pi. Hamsters were monitored for appearance, behavior and weight. At day 4 pi, hamsters were euthanized by i.p. injection of 500 μL Dolethal . Lungs were collected and viral RNA and infectious virus were quantified by RT-qPCR and end-point virus titration. Two independent studies were performed each with a total 24. Standards of SARS-CoV-2 cDNA (IDT) were used to express viral genome copies per mg tissue23.Hamster lung tissues were collected after sacrifice and were homogenized using bead disruption (Precellys) in 350 µL TRK lysis buffer  and centrifuged  to pellet the cell debris. RNA was extracted according to the manufacturer’s instructions. RT-qPCR was performed on a LightCycler96 platform (Roche) using the iTaq Universal Probes One-Step RT-qPCR kit (BioRad) with IDT nCOV_N2 primers and probes targeting the nucleocapsid 25 using the Lindenbach calculator and were expressed as 50% tissue culture infectious dose (TCID50) per mg tissue.Lung tissues were homogenized using bead disruption (Precellys) in 350 µL minimal essential medium and centrifuged  to pellet the cell debris. To quantify infectious SARS-CoV-2 particles, endpoint titrations were performed on confluent Vero E6 cells in 96- well plates. Viral titers were calculated by the Reed and Muench methodFor histological examination, the lungs were fixed overnight in 4% formaldehyde and embedded in paraffin. Tissue sections (5 μm) were analyzed after staining with hematoxylin and eosin and scored blindly for lung damage by an expert pathologist. The scored parameters, to which a cumulative score of 1–3 was attributed, were the following: congestion, intra-alveolar hemorrhagic, apoptotic bodies in bronchus wall, necrotizing bronchiolitis, perivascular edema, bronchopneumonia, perivascular inflammation, peribronchial inflammation and vasculitis.The viral RNAs isolated from the lungs of infected animals in the transmission study and the nirmatrelvir-treated animals in the treatment study were subjected to deep sequencing analysis of the full genome sequence through ARTIC SARS-CoV-2 RNA-Seq service provided by Eurofins Genomics. Alignment of the obtained sequences was performed using the Geneious Prime software.The detailed statistical comparisons, the number of animals and independent experiments that were performed is indicated in the legends to figures. “Independent experiments” means that experiments were repeated separately on different days. The analysis of histopathology was done blindly. All statistical analyses were performed using GraphPad Prism 9 software . Statistical significance was determined using the non-parametric Mann Whitney U-test. P-values of <0.05 were considered significant.Housing conditions and experimental procedures were done with the approval and under the guidelines of the ethics committee of animal experimentation of KU Leuven (license P065-2020).Further information on research design is available in the Supplementary InformationPeer Review FileReporting Summary