Treating this viral outbreak like COVID-19 should be discouraged. In May 2022, the world witnessed the re-emergence of the zoonotic disease monkeypox. While this was not the first epidemic of this disease, what differentiated the outbreak was the rapid global spread and increase of cases, which led the WHO to declare monkeypox a global health emergency. Although the disease spreads mainly through inadequately cooked meat of various rodent species, this virus also shows droplet, respiratory, sexual, and even vertical transmission. Monkeypox further multiplies in lymphoproliferative organs and presents with a classical smallpox-like rash, fever, headache, and muscle aches. Diagnosis is confirmed with a polymerase-chain-reaction test and is managed largely supportively with possible usage of some antivirals and immunoglobulins. Moreover, some pre-exposure and postexposure prophylactic vaccines have been developed. This paper aims to conduct an in-depth review of the historical epidemics, transmission, pathophysiology, clinical presentation, and management of the monkeypox disease.  Although an outbreak was ongoing in Nigeria and other African countries since 2017, the disease seemed to be limited to previously endemic areas However, following the virus's first detection in the UK, several countries have reported new cases and, as of the 22nd of September (according to the US Centers for Disease Control and Prevention (CDC)), approximately 65,415 cases and 26 deaths have been registered in 106 countries, 99 of which had no previously historically registered cases of the disease  [2]. DesOrthopoxvirus genus, the same genus as smallpox with whom it shares some similarities, despite being less severe. Although scientists first identified the virus in laboratory monkeys in 1958, the first-ever human infection was documented in 1970 in the Democratic Republic of Congo Monkeypox is a zoonotic disease caused by the monkeypox virus (MPXV), a double-stranded DNA virus belonging to the While the literature has covered different aspects of monkeypox since the onset of the outbreak, a review that addresses the disease in a comprehensive way is lacking. This article aims at serving as an integrative, comprehensive review of the most recent monkeypox outbreak, providing a detailed insight into the genomics of the causative virus, the historical background of previous outbreaks, and highlighting the available treatment and prevention measures as well as the principal public health strategies implemented to control the spread of the disease.2Monkeypox virus was first isolated in 1958 in Copenhagen from a group of laboratory cynomolgus monkeys used for researching poliovirus vaccines after one of the monkeys had developed pox-like skin cutaneous lesions The first human infection with MPXV was documented in 1970 in a 9-month-old boy in the Democratic Republic of Congo (DRC), previously known as Zaire Further outbreaks were reported in Africa in the years 1981–1986 and 1996–1997, characterized by a lower case-fatality ratio and a greater attack rate than typical outbreaks. A total of 986 cases and 6 deaths were recorded in the continent during that period which was considered the largest outbreak since virus isolation In 2003, the virus made its first appearance outside of Africa in the United States. A total of 71 cases were reported by the CDC in Wisconsin (39), Indiana (16), Illinois (12), Missouri (2), Kansas (1), and Ohio (1) In 2005, concerns were raised following reporting of a case in southern Sudan, which represented the first African case outside the Congo Basin and western African regions In September 2017, Nigeria witnessed a re-emergence of the disease after almost 4 decades of latency May 2022 registered a surge in cases in nonendemic areas of the world, which prompted the global healthcare authorities to take necessary action to contain the spread of the virus 3Orthopoxvirus genus, Poxviridae family, and Chordopoxvirinae subfamily  and extracellular enveloped virus (EEV) to the surface of the target cell. VV–A28, one of the particles’ virion proteins, mediates the virus fusion and release of its core structure into the cytoplasm of the infected cell. The first pathway of early gene expression that leads to viral mRNA synthesis begins under endogenous RNA polymerase influence. The start of the intermediate and late transcription phases is marked by the release of viral DNA into the host's cytoplasm to act as a template for viral replication through a process known as uncoating. This is followed by the assembly of virion particles to form IMV which leads to the formation of intracellular enveloped virus (IEV) which fuses with the cell membrane to form the cell-associated enveloped virus (CEV). While CEV and EEV are thought to be implicated in early cell-to-cell spread, IMV aids viral spread at later stages 3.2After entering the human body via the airways, the oral cavity, or infected body fluids ingestion, the virus travels to a local lymph node or through the blood, causing primary viremia. It then travels to distant lymph nodes causing lymphadenopathy by enhancing the proliferation of natural killer cells and entering the systemic circulation through the lymphoproliferative organs, causing secondary viremia 3.3Poxviruses exhibit 3 types of viral tropism which play an important role in determining the ability of the virus to infect various host species. The first type, cellular tropism, indicates the different modalities in which the virus replicates in infected species. The second type, known as tissue tropism, refers to the increased viral replication in particular hosts or organs mediated by specific antiviral responses, whereas the third type, known as organism tropism, refers to the pathogenesis and clinical picture of the virus under the effects of host's immune responses. The combination of the last 2 types of tropism determines the host-to-host transmission of the virus 3.4Although recent instances of MPXV infection were all phylogenetically connected to the West African clade, at least 2 distinct clades have been found, each with a different fatality rate Orthopoxvirus orthologs is lost in MOPICE from Congo Basin isolates due to a C-terminal truncation The Congo Basin isolates have the monkeypox homolog of SPICE (monkeypox inhibitor of complement enzymes (MOPICE)), while the sequenced West African viruses do not Members of the SPICE family are candidates for virulence and transmissibility factors linked to human orthopoxvirus disease due to the presence of MOPICE in the more virulent and transmissible Congo Basin MPXV isolates and SPICE in variola, as well as the absence of MOPICE in the less virulent West-African monkeypox isolates. Candidate open reading frames D10L (host range), B10R (virulence factor for myxoma virus), B14R (interleukin (IL)-1 binding protein), and B19R (serine protease inhibitor-1) are further candidates that may be significant for the virulence and transmissibility of Congo Basin MPXV isolates 3.5+ and CD8+ T cells, therefore, avoiding the host's systemic immune suppression Numerous mechanisms have been developed by orthopoxviruses in general and monkeypox in specific to evade the host's immune response 3.6Orthopoxvirus genus that is known to infect humans and animals The West African clade of MPXV is believed to have been present for the past 600 years, and it is an important member of the The recent 2022 MPXV outbreak is no exception to that, except that it has gone under the radar for long enough in low-and-middle-income countries. In a study performed by João Paulo Gomes et al., the genomes of the MPXV in 15 infected individuals during the 2022 outbreak were compared. The patients had distinct strains of monkeypox, which the researchers were able to link to an earlier outbreak of the virus that occurred in 2018–2019 in the United Kingdom, Israel, and Singapore and had its origins in Nigeria According to another study by the UK Health Security Agency (UKHSA), in comparison to the UK MPXV from 2018, the strain of the 2022 outbreak had 48 unique changes in its genome. Twenty-seven of these mutations were silent, meaning they did not affect the viral proteins while 21 of them altered viral proteins in some way Based on the known function of the viral proteins, mutations were divided into 3 categories by the UKHSA assessment: low, medium, and high priority for inquiry . These e3.7Poxviridae and the genus Orthopoxvirus. According to comparative genomics, all current orthopoxvirus species originated from a single ancestor through a process that was characterized by the loss of a group of accessory genes that was specific to each lineage. The original virus is thought to resemble specific cowpox virus strains, which have a wide range of hosts and almost the entire collection of accessory genes Monkeypox viral evolution is convoluted. The virus is a member of the family The variola  virus and the MPXV are closely related, and both affect people with a febrile rash sickness that is like but less severe than smallpox. According to a study published by Douglass and Dumbell, despite having several deletions, a sequence in the DNA of the MPXV exists that is a homolog of a 1,065-bp open reading frame in the conserved area of the variola virus genome The evolution of zoonotic infections to become more contagious or virulent in humans is a huge concern. An in-depth analysis published by Isidro et al. strongly suggests an ongoing evolution of the MPXV. It used Shotgun metagenomics to quickly reconstruct and characterize the first MPXV outbreak genome sequences, demonstrating that this current MPXV belongs to clade 3 and that the outbreak most likely originated from a single source. Despite clustering with cases from 2018–2019 that were connected to a nation where the virus is endemic, 2022 MPXV (lineage B.1) segregates in a divergent phylogenetic branch, most likely suggesting ongoing accelerated evolution. A comprehensive examination of mutations points to the role played by the host APOBEC3 in viral evolution as well as possible MPXV human adaptations in continuing microevolution. The findings also suggest that genome sequencing might be able to help track the transmission and spread of this double-stranded DNA virus Moreover, MPXV is likely to evolve slowly as its DNA genome replicates by a viral DNA polymerase with 3′–5′ exonuclease proofreading activity, which has a lower mutation rate than RNA viruses All the above investigations suggest that further studies are required to establish the origin, evolution, and ongoing development of the MPXV.3.8β-binding protein (IL-1) that is considered responsible for the attenuation of the disease caused by the virus Monkeypox has been long thought to have evolved from smallpox . Genetic4The identified mode of transmission of MPXV in Africa is typically a zoonosis involving living in forested or deforested areas, handling or consuming products of infected meats, and contacting animals or their bodily fluids 4.1The WHO has confirmed that MPXV spreads from human to human, mainly through close contact with respiratory secretions of infected people, skin lesions, or objects contaminated with bodily fluids or diseased tissue of infected people, and through respiratory droplets Forty-seven infected patients from the prairie dogs demonstrated high susceptibility of prairie dogs to MPXV 5With increasing travel worldwide and the long incubation period, it is unsurprising that MPXV cases have been reported in nonendemic nations. The recent monkeypox outbreak which affected many MSM raised concerns about possible sexual transmission A group of researchers further investigated sexual transmission and concluded that MPXV can be transmitted through bodily fluids and close skin-to-skin contact from active lesions. The clinical manifestation is generally described as mild, with most lesions on the genitalia or peri-genital areas, indicating that transmission likely occurred during close physical contact during sexual activities. These were believed to be the first cases of MPXV infection with documented transmission through sex 5.1Possible vertical transmission was hypothesized by many researchers before. There were also cases of vertical transmission that led to pregnancy loss due to fetal infection In previous outbreaks, we had 4 cases of reported MPXV infection during pregnancy in the Democratic Republic of Congo (DRC), where one patient had mild disease and no subsequent pregnancy-related complications. However, 2 and one of the 3 other women had a first and second-trimester pregnancy loss, respectively. The second-trimester fetus had apparent hydrops, hepatomegaly, vesicular rash, and confirmed MPXV in its blood using PCR Therefore, it is important to consider close fetal monitoring in pregnant women with confirmed MPXV infection. Delivery with cesarean section can be an option 5.2Evidence of MPXV infection has been found in various animal species in Africa, including rope squirrels, tree squirrels, Gambian pouched rats, dormice, nonhuman primates, and other species Poxviridae to new animal hosts has occurred previously. The 2003 US MPXV outbreak added to the breadth of host species capable of supporting MPXV replication and paid attention to the potential for MPXV to expand its geographic range The adaption of Under laboratory or captive conditions, a broad range of mammalian taxa was likely susceptible to MPXV infection 5.3Symptoms of monkeypox resemble those of smallpox in humans and begin with fever, headache, muscle aches, and exhaustion . The incInfection can be divided into 2 phases: the initial invasion period (lasting from 0–5 days) which manifests as high fever, intense headache, lymphadenopathy, back pain, myalgia, and exhaustion, and the skin rash period (appearing within 1−3 days from the onset of fever). The rash occurs more on the face and extremities but can also involve the oral mucous membranes, genitalia, conjunctiva, and cornea. Despite the similarities in the clinical picture, smallpox, and monkeypox differ primarily in that monkeypox causes lymph node enlargement (lymphadenopathy), whereas smallpox does not Monkeypox is usually a self-limiting disease. A person is contagious from the development of enanthem till the crusts fall off. Severe cases and worse outcomes commonly occur in children and individuals with underlying immune deficiencies.5.4A study across 16 countries showed a characteristic rash in 95% of patients , with over 64% having more than 10 lesions present. The vesiculopustular lesions are deep and well-circumscribed with a characteristic umbilication with all lesions being of similar size, and at various stages across the site. Some cases of a single genital ulcer or lesion were also seen and warrant the possibility of misdiagnosis as a sexually transmitted infection 5.5The current prodromal symptoms are seen to appear after the rash and have only been seen in around half of the cases, which again suggests the unusual nature of this 2022 outbreak 5.6Most cases of monkeypox currently seen in children are a result of household contact; however, the susceptibility and clinical outcomes are still unknown. Evidence shows increased severity in children younger than 8 years, children with underlying immune deficiencies, and children with pre-existing skin conditions. The presentation seen in children mimics the atypical pattern seen in adults with a characteristic rash that may or may not be accompanied by other symptoms. This rash may be misdiagnosed as other rashes commonly seen in children such as varicella, measles, scabies, allergic eruptions, etc. Complications are rare but include encephalitis, cellulitis, pneumonia, sepsis, abscess, and lymphadenopathy resulting in airway obstruction, keratitis, and corneal scarring 6The factors that need to be considered in the diagnosis of monkeypox are the date of onset of fever and rash, recent travel history to endemic areas within 21 days preceding the onset of the symptoms, history of contact with a suspected infected person, date of specimen collection, and history of vaccination against smallpox. For clinical diagnosis, other rash illnesses like chickenpox, measles, bacterial skin infections, scabies, and syphilis must be excluded 7Infection from MPXV is milder as compared to smallpox and is usually self-limiting, resolving in about 2–4 weeks with bed rest and supportive care. Rarely, cases may develop severe dehydration, pitted and deforming scars, secondary bacterial infection, bronchopneumonia, respiratory distress, ocular infection, septicemia, and encephalitis. Management of the disease includes supportive treatment, antivirals, and immunoglobulins. Hospitalization in a negative pressure room is generally preferred for the management of complications 7.1The lack of effective antiviral drugs against MPXV, combined with the spontaneous recovery of most of the infected patients without the need for medical treatment 7.2Orthopoxvirus species. The former is an intravenously administered drug that inhibits viral DNA polymerases, whereas the latter is an orally administered modified cidofovir that has a better safety profile and is less nephrotoxic Cidofovir and brincidofovir (CMX-001) are antiviral agents with activity against Orthopoxvirus infection in humans For the current outbreak, tecovirimat and brincidofovir have been approved in the US to be used against monkeypox Tecovirimat acts by inhibiting the VP37 viral envelope protein, which limits the last steps in viral maturation and release from the infected cell, preventing virus dissemination inside an infected host Brincidofovir  is a lipid conjugate of cidofovir, an acyclic nucleoside analog approved for the treatment of human cytomegalovirus retinitis in AIDS patients 8Smallpox vaccines are at least 85% cross-protective against monkeypox. Pre-exposure prophylaxis against the virus can be achieved by various types of vaccines: a live-attenuated vaccinia vaccine (ACAM2000), a nonreplicating modified vaccinia Ankara (MVA), JYNNEOS, and a minimally replicating vaccine (LC16). Postexposure vaccination in unvaccinated individuals exposed to the disease effectively prevents future infections and, if given within 3 days of the exposure, can reduce the severity of the symptoms. JYNNEOS was shown to protect against the side effects documented in earlier vaccines. Nevertheless, the 28-day gap between the first and second dose may decrease its protective value in postexposure prophylaxis. Achieving mucosal immunity may play a pivotal role in preventing disease transmission, being mucous membranes a route by which the virus spreads. The ability of the available vaccines to produce mucosal immunity, however, is not yet confirmed 8.1Vaccinia Immune Globulin Intravenous (VIGIV) are antibodies obtained from the pooled blood of smallpox-vaccinated individuals. Although no clinical data is available on the efficacy of VIGIV for the management of monkeypox complications, it can be considered in combination with antivirals for severe infection. Moreover, the therapy is safe and may help treat vulnerable patients Moreover, a study conducted by Stittelaar et al. concluded that “during an outbreak, cidofovir or other related nucleotide analogs should be considered as an additional strategy for vaccination of individuals exposed to smallpox or other poxviruses/orthopoxviruses, including monkeypox” 8.2MVA-BN vaccine, a third-generation live vaccine that is nonreplicable in humans 9The key to preventing monkeypox is increasing public awareness of risk factors and minimizing exposure to viruses. According to the CDC's recommendations, any person exhibiting symptoms and signs should undergo complete investigations for confirmation of monkeypox. If confirmed, isolation and contact tracing are recommended to decrease the spread of the virus. Furthermore, contact with animals that might act as a reservoir of the virus should be avoided. In case of contact, hands should be immediately sanitized. According to WHO, vaccination against smallpox was demonstrated through several observational studies to be about 85% effective in preventing monkeypox 1010.1Monkeypox patients should be isolated at home until the rash has completely disappeared. This is because infection from the nonintact skin is highest in monkeypox. Having visitations, close contact with people and animals, or sexual activity are high-risk behaviors that can lead to the rapid transmission of the disease. Patients should not share any materials  with other household members. A separate bathroom is favorable, but if that is not feasible, disinfecting the bathroom after usage with alcohol-based materials should be done. Proper hand hygiene and wearing gloves and face masks should be exercised regularly if contact is imminent. The CDC also recommends wearing tall clothes covering most of the body area 10.2Because monkeypox is mostly spread through physical contact, the following activities are advised in hospitals and clinics besides standard precautions applied to all patients. Monkeypox patients should adhere to transmission-based precautions, specifically contact precautions, which include isolating them in a separate room, cleaning and disinfecting the room at least daily, performing proper hand hygiene techniques with soap and water or using an alcohol-based hand sanitizer, limiting transport and movement of patients outside the room unless for medically-necessary purposes, and providing individual patient care equipment such as stethoscopes and blood pressure cuffs. Healthcare workers should use personal protective equipment (PPE) before entering the patient's room. This typically includes wearing gloves, gown, eye protection, and NIOSH-approved particulate respiratory equipped with at least N95 filters for all interactions. Removing PPE before leaving the room, performing a hand hygiene routine immediately, and ensuring that no surfaces or objects in the patient's room are touched after the removal of PPE are also required measures to ensure the safety of healthcare personnel and other patients in the hospital 10.3The amount of pox virus that is expected to be present in clinical samples of blood and bodily fluids is minimal. Because of this, immunization is not advised for staff who handle and analyze common clinical specimens from monkeypox patients , chemistries, and microbiology). It is advised to use biosafety level 2 (BSL-2) confinement to take general precautions to safeguard against any potential infectious pathogens present in any specimen received. The MPXV will not be present in clinical specimens if standard precautions and biosafety measures are consistently followed for the protection of laboratory personnel. Specimen testing should only be performed by a small number of employees, and processes that could produce infectious aerosols should be avoided 10.4All wastes collected from the patient should be promptly inactivated on-site or safely sent to a special facility that is specialized in dealing with Category A waste of infectious substances, according to the US Department of Transportation (DOT) 10.5The main goals of surveillance and case investigation for monkeypox are to quickly identify cases, clusters, and the sources of infection to provide the best clinical care, isolate cases to stop further transmission, identify and manage contacts, and develop efficient control and prevention strategies based on the most frequently discovered routes of transmission. According to the WHO, the definition of contact has had one more of the following exposures with a probable or confirmed case of monkeypox: face-to-face exposure , direct physical contact, including sexual contact, contact with contaminated materials such as clothing or bedding. For 21 days after the last contact with a patient or their contaminated materials during the infectious period, contacts should be checked at least once a day for the appearance of signs or symptoms 11During the most recent outbreak, it has been revealed that the virus was spreading undetected in various African countries where multiple outbreaks have happened before. However, before the spread to the Western world, there were no statistics on how serious the problem was, except for the report published by the WHO, stating that Cameroon, Central African Republic, Congo, and Nigeria have reported several monkeypox cases  and deaths  in early 2022. Furthermore, the DRC has experienced a surge in the number of monkeypox cases since March 2019. Surprisingly, the investigation for this outbreak started 21 months later (January 2021), and that is when the public health team identified 1,238 cases and 57 deaths in 2022 12This review serves to provide a comprehensive understanding of MPXV virology, genomic structure, transmission, diagnosis, management, and infection control measures. Despite the numerous unknowns about the recent monkeypox outbreak, its mild clinical picture makes it easier to treat. Still, implementing wide public health measures is important to hinder the spread of the virus, although its potential to progress into a pandemic is unlikely. Although the certain reason behind the sudden re-emergence of the virus is unknown, one of the strongest hypotheses is the decrease in smallpox vaccination coverage following its eradication. Nevertheless, haphazardly injecting the populations with smallpox or monkeypox vaccines should be discouraged. The fear of a “new COVID-19” shall not hinder our scientific judgment in dealing with future monkeypox outbreaks the right way and care should be taken in avoiding the shortcomings that happened during the COVID-19 outbreak. A thorough investigation of the reasons that led to the virus going undetected for a long time in LMICs is paramount in tackling the threat of future outbreaks. Finally, the adoption of a One Health approach for the timely detection and response to re-emerging zoonotic infections is essential.R.E.: Conceptualization, Data curation, Visualization, Methodology, Project administration, Resources, Writing – original draft. A.M.M.: Conceptualization, Data curation, Visualization, Methodology, Project administration, Resources, Writing – original draft. T.V.: Conceptualization, Writing – original draft. S.T.: Writing – original draft. K.S.: Writing – original draft. N.D.T.: Writing – original draft. S.S.E.: Writing – original draft. N.T.V.: Writing – original draft. N.T.H.: Conceptualization, Data curation, Project administration.


Understanding how to harden liquid condensates produced by influenza A virus could accelerate the development of novel antiviral drugs. Related research article Etibor TA, Vale-Costa S, Sridharan S, Brás D, Becher I, Mello VH, Ferreira F, Alenquer M, Savitski MM, Amorim MJ. 2023. Defining basic rules for hardening influenza A virus liquid condensates. eLife12:e85182. doi: 10.7554/eLife.85182.Understanding how viruses infect cells, replicate, and subsequently spread through the body is crucial for developing effective antiviral therapies. During this process, most common viruses – including the one responsible for flu, influenza A virus – form membrane-less organelles called condensates which help the virus to assemble its genome and replicate . While sInfluenza A virus is thought to induce condensates in order to help with genome assembly . Its genThe team  monitored the structure and orientation of viral inclusions by measuring their number, shape, size, and density. How the inclusions moved and interacted with each other was also studied through live-cell imaging and by calculating their molecular stability . Etibor The experiments revealed that while changes in temperature and the concentration of vRNPs and Rab11a altered the size of the viral inclusions, the material properties of the inclusions remained mostly the same. These results are surprising as previous studies have shown that, in general, condensates strongly depend on these two factors. Etibor et al. noted that these findings may be specific to influenza A virus, as its condensates need to maintain liquid-like properties over a wide range of vRNP concentrations to replicate efficiently.Next, Etibor et al. treated cells with nucleozin, a pharmacological modulator that has been shown to lower the viral load in patients with influenza A in preclinical studies . NucleozThe team also showed that nucleozin stiffened viral inclusions in the lung cells of mice infected with influenza A virus, and helped speed up the mice’s recovery. Furthermore, nucleozin did not alter the level of other proteins in the cells of the mice, demonstrating the drug’s specificity against the virus.In summary, Etibor et al. revealed how different factors influence the material properties of viral inclusions in both cells and mice infected with influenza A virus. Their findings suggest that stabilizing vRNP interactions shows the most promise for disrupting the function of viral inclusions, and highlight the potential of antiviral drugs that harden these condensates.


Is higher resource utilization, quantified by hospital costs, associated with survival without neurodevelopmental impairment among infants with hypoxic-ischemic encephalopathy treated with therapeutic hypothermia?In this cohort study of 381 neonates with hypoxic-ischemic encephalopathy who underwent therapeutic hypothermia in 11 children’s hospitals, total hospitalization costs during the first 4 days were not associated with neurodevelopmental outcomes. Higher electroencephalography (EEG) costs were associated with survival without neurodevelopmental impairment, yet higher antiseizure medication or laboratory costs were not.These findings suggest EEG monitoring may improve outcomes, whereas more intensive laboratory monitoring may not yield similar benefits. This cohort study examines whether there is an association between higher resource utilization during the first 4 days of age, quantified by hospital costs, and survival without neurodevelopmental impairment among infants with hypoxic-ischemic encephalopathy (HIE). Intercenter variation exists in the management of hypoxic-ischemic encephalopathy (HIE). It is unclear whether increased resource utilization translates into improved neurodevelopmental outcomes.To determine if higher resource utilization during the first 4 days of age, quantified by hospital costs, is associated with survival without neurodevelopmental impairment (NDI) among infants with HIE.Retrospective cohort analysis of neonates with HIE who underwent therapeutic hypothermia (TH) at US children’s hospitals participating in the Children’s Hospitals Neonatal Database between 2010 and 2016. Data were analyzed from December 2021 to December 2022.Infants who survived to 4 days of age and had neurodevelopmental outcomes assessed at greater than 11 months of age were divided into 2 groups: (1) death or NDI and (2) survived without NDI. Resource utilization was defined as costs of hospitalization including neonatal neurocritical care (NNCC). Data were linked with Pediatric Health Information Systems to quantify standardized costs by terciles.The main outcome was death or NDI. Characteristics, outcomes, hospitalization, and NNCC costs were compared.P < .001) and a lower rate of infants with mild or moderate HIE  compared with the survived without NDI group. Compared with low-cost centers, there was no association between high– or medium–hospitalization cost centers and death or NDI. High– and medium–EEG cost centers had lower odds of death or NDI compared with low-cost centers . High– and medium–laboratory cost centers had higher odds of death or NDI compared with low-cost centers . High–antiseizure medication cost centers had higher odds of death or NDI compared with low-cost centers .Among the 381 patients who were included, median (IQR) gestational age was 39 (38-40) weeks; maternal race included 79 (20.7%) Black mothers, 237 (62.2%) White mothers, and 58 (15.2%) mothers with other race; 80 (21%) died, 64 (17%) survived with NDI (combined death or NDI group: 144 patients [38%]), and 237 (62%) survived without NDI. The combined death or NDI group had a higher rate of infants with Apgar score at 10 minutes less than or equal to 5 (65.3% [94 of 144] vs 39.7% [94 of 237]; Hospitalization costs during the first 4 days of age in neonates with HIE treated with TH were not associated with neurodevelopmental outcomes. Higher EEG costs were associated with lower odds of death or NDI yet higher laboratory and antiseizure medication costs were not. These findings serve as first steps toward identifying aspects of NNCC that are associated with outcomes. EEG use may vary by center depending on availability of full montage video EEG (vEEG) or amplitude integrated EEG (aEEG), and the presence of technicians and electrophysiologists. Similarly, we previously reported significant intercenter variation with regards to ASM use in HIE.7 Utilization of ASM may vary based on center guidelines , use of continuous EEG to identify electrographic seizures, and/or individual clinician practice.Laboratory monitoring, electroencephalography (EEG), and antiseizure medications (ASM), which are central to contemporary neonatal neurocritical care (NNCC) during the first 4 days of admission for HIE, are variably used among centers.9 it remains unclear whether increased (or decreased) resource use is associated with improved long-term outcomes. We sought to determine if higher resource utilization, measured by standardized costs during the first 4 days of age in the neonatal intensive care unit (NICU), a period corresponding to TH and rewarming, is associated with survival without neurodevelopmental impairment (NDI) among infants with perinatal HIE. A secondary objective was to determine if specific NNCC costs  are associated with outcomes.Although there is wide intercenter variation with regards to resource utilization in HIE,STROBE) reporting guideline. The institutional review board (IRB) at each participating institution approved participation in the Children’s Hospitals Neonatal Database (CHND) and additional IRB approval was obtained at centers participating in this study to collect developmental follow-up data. Waiver of consent was granted by the IRBs at the 11 participating centers as this was a minimal risk retrospective database study.This is an analysis of linked data from 3 multicenter data sources described later from neonates with HIE treated at 11 regional US NICUs participating in the Children’s Hospitals Neonatal Consortium. Cost was used as an indirect measure of resource utilization during the first 4 days of age for HIE admission and for ascertaining use of specific NNCC components such as laboratory, EEG, and ASM. This study follows the Strengthening the Reporting of Observational Studies in Epidemiology  contains detailed hospital administrative and billing data from more than 40 pediatric institutions.2 Methods ensuring data quality for both databases have been reported.11 Developmental outcome data were retrospectively collected at individual participating sites.12 CHND, PHIS, and developmental outcome data were linked at the patient level using unique identifiers unavailable to investigators.The CHND prospectively captures detailed clinical data from all infants admitted to participating level IV NICUs.8 was queried to identify neonates born at participating centers between July 2010 and July 2016 with the diagnosis of perinatal HIE according to established criteria,5 treated with TH, admitted less than 2 days of age, at least 36 weeks’ gestation and 1800 g at birth. Neonates were excluded if they had major congenital anomalies, if linkage to PHIS was not possible, or if they did not survive through the first 4 days of age. Survivors were excluded if neurodevelopmental assessments were not available at greater than 11 months of age. Maternal race and ethnicity data were collected as reported from the CHND; these data were collected to assess if these factors were associated with outcome. Race and ethnicity groups included were White, Black, or other .The CHND13 Briefly, costs for every CTC billing code were computed based on a ratio of cost to charge and adjusted for geographical region wage and price index. A standardized unit cost for each CTC code was defined as the median of unit costs across all PHIS-participating hospitals and tabulated in a cost master index (CMI) as previously described.13 The CMI-based standardized cost was used to estimate hospitalization costs for every admission in the study cohort. Total cost of hospitalization was calculated per patient and per center for the first 4 days, to allow for evaluation of patient- and/or clinician-level and center-level variation as potential risk factors of outcome. Analogous costs were also calculated for laboratory tests, EEG, and ASMs during this study period.PHIS Clinical Transaction Classification (CTC) codes corresponding to hospital billing for admission and any type of laboratory test, EEG, and ASM charge were used to quantify standardized costs as a reflection of frequency and duration of resource use. Standardized costs were calculated according to a previously described cost master index.14 For participants assessed using the Capute Scales or the Ireton Infant Development Inventory, NDI was defined as a developmental quotient less than 70.17 These definitions are similar to moderate or severe disability used in HIE cooling trials.18Because all sites in the consortium did not use the same developmental assessments, we defined NDI at greater than 11 months of age if the participant had any of the following: substantial developmental delay (defined based on the specific assessment tool used), diagnosis of cerebral palsy  score ≥2), or presence of deafness (requiring amplification) or blindness. For participants assessed using the Bayley Scales of Infant Development, Third Edition (BSID-III), NDI was defined by a BSID-III composite cognitive score less than 85 or a composite motor score less than 70.P < .05. Data were analyzed from December 2021 to December 2022 using SAS Enterprise Guide version 7.1 (SAS Institute).19Study sample size was based on a convenience sample of consecutive admissions of infants meeting inclusion criteria during the study period. Study population characteristics and cost distribution data were described using standard summary statistics. Cost data were log transformed to account for the skewed distribution. Costs were categorized as low, medium, and high based on thresholds derived from calculating terciles at both the individual case and center levels. Univariable and multivariable logistic regression analyses were performed to assess the associations between costs and outcome groups. Analyses were adjusted for covariates including HIE severity, EEG confirmed seizures, inhaled nitric oxide (iNO), extracorporeal membrane oxygenation (ECMO), ventilator days and socioeconomic status . To evaluate the association between center EEG and ASM costs, the 25th and 75th percentile of the center median EEG costs were plotted on the vertical axis and of the median center ASM costs on the horizontal axis. The significance level was set at 2-sided A total of 1693 patients with HIE were entered into the CHND during the study period. Of the 574 patients that had outcome measures, PHIS linkage was unavailable for 128 patients and 65 patients died within the first 4 days of age. The final analysis included 381 patients; of these, 80 (21%) died, 64 (17%) survived with NDI, and 237 (62%) survived without NDI ; median P < .001), be born at home  and less likely to have mild to moderate HIE   . Rates oP < .001), received iNO , and had higher median (IQR) ventilator time  than infants who survived without NDI. The death or NDI group had a shorter median LOS . The need for ECMO did not differ between groups . Among NNCC costs, laboratory costs were highest, accounting for 14% of total costs, followed by EEG, accounting for 9% of total costs. Laboratory costs and ASM costs were higher in the death or NDI group. EEG costs were higher in the survived without NDI group (Costs and comparisons between groups are presented in DI group . UnadjusDI group  and loweDI group .P = .55) (P < .001) (P = .02) (P = .007)  . High– a < .001) . High– (P = .02) . Compare = .007) . We did  = .007) .P = .51) (P = .001)  or medium–hospitalization costs  and death or NDI (P = .51) . High– ( = .001) . EEG and = .001) .In neonates with HIE treated with TH at regional children’s hospitals, total hospitalization costs during the first 4 days of age were not associated with risk for death or NDI. Meanwhile, infants cared for at high–EEG cost centers had lower odds of death or NDI despite controlling for differences in clinical severity and medical comorbidities. Conversely, we found that higher laboratory costs and ASM costs were not associated with improved survival without NDI.1 however, components of its associated NNCC have not been fully studied. We previously reported the median (IQR) hospitalization cost per case among 19 NICUs participating in CHND for survivors was $58 552 ($32 476-$130 203) and for nonsurvivors was $29 760 ($16 897-$61 399).2 Center differences accounted for 29% of variation in total hospitalization cost with the widest cost variability across centers being EEG.2 The Canadian Neonatal Network reported practice and outcome variations in neonates with HIE treated with TH across Canada.9 Practice differences in this report were mainly in the use of inotropes, iNO, blood products, and feeding during TH. The rates of mortality and brain injury also varied significantly across NICUs. None of these prior reports evaluated whether costs or resource utilization translated into improved neurodevelopmental outcomes.TH is a cost-effective strategy in the management of neonates with moderate to severe HIE,2 We have also observed the median age of death in neonates with HIE is 4 days, with the majority occurring after withdrawal of life sustaining support.20 Therefore, while using a cutoff of death greater than 4 days excluded 45% of the patients who died, we found that baseline characteristics were similar between deaths within 4 and after 4 days. Additionally, those that died within 4 days had lower costs which would have skewed our cost quantification due to unequal observation period. Although unadjusted hospitalization costs were significantly higher for the death or NDI group, it was reassuring to find that adjusted hospitalization costs were not associated with long-term disease burden.Variations in costs can occur at the patient and center level, therefore, we ranked costs to account for clinician variation as well as center-specific practices. We did not find hospitalization costs during the first 4 days of age, at center nor case level to be associated with outcomes. We focused on more than 4 days of age as our prior work showed that hospital costs are associated with length of stay.24 In neonates with HIE, seizures are a manifestation of underlying brain injury and high seizure burden is associated with severity of encephalopathy, more severe brain injury, and worse developmental outcomes in survivors of HIE.27 Kharoshayanka et al25 noted more than 9-fold higher odds of poor outcome if seizure burden exceeded 40 minutes and an 8-fold increase if hourly seizure burden was greater than 13 minutes per hour. Early detection, identification and treatment of seizures could therefore potentially improve outcomes. However, neonatal seizures are often subclinical and difficult to identify even for experienced clinicians.28 Continuous EEG monitoring allows earlier detection of electrographic seizures, reduces the time to ASM therapy and decreases seizure burden.31 A recent study by Hunt et al32 in contrast did not reveal such utility or benefit to using aEEG monitoring to direct treatment of electrographic seizures with regards to death or disability at 2 years of age. However, the analyses did not account for differences in ASM use or seizure severity and/or burden.32 Although our study showed an association between higher EEG costs and improved outcomes, we were not able to account for aEEG use as the analysis was limited to CTC codes for EEG hospital charges.The association between higher center EEG costs and improved outcomes is an interesting finding that suggests further study may indicate possible practice changes. Preclinical animal studies suggest that seizures are not just a manifestation of brain injury but themselves cause additional brain injury, behavioral deficits, and age-dependent impairment of neurogenesis.33 evaluated their single center data during 3 time periods of changing EEG monitoring practices . They found a higher sensitivity and specificity of cEEG for seizure detection, and fewer infants prescribed ASM when undergoing aEEG (38% reduction) and cEEG (67% reduction) compared to single EEG use alone.33 This suggests that EEG monitoring, specifically cEEG monitoring, may be helpful to avoid overtreatment. It is reassuring that in our study, center median EEG costs did not correlate with higher ASM costs. It is possible that higher EEG costs (correlated with use) may be associated with lower ASM costs per use, however, this needs to be further investigated. We also found that higher ASM cost was associated with higher odds of death or NDI at the center but not case level, after controlling for HIE severity and presence of EEG seizures. Animal studies have shown that ASMs cause cell death, however, human studies are limited.35 Our study findings suggest that center-driven practices such as increased EEG monitoring may play a role in improving neurodevelopmental outcomes in neonates with HIE, and are consistent with neuromonitoring guidelines proposed by the American Clinical Neurophysiology Society36 and Newborn Brain Society.37EEG monitoring could limit or reduce unnecessary ASM use, for example, by identifying paroxysmal events without electrographic correlate that can be mistaken for seizures. Jan et al38 Although the higher laboratory costs may be reflective of the severity of medical illness which may correlate with risk for adverse neurologic outcomes, our analyses were adjusted for medical comorbidities such as iNO/ECMO and need for mechanical ventilation.It is important to note that higher laboratory costs at the center and case level, indicative of more intensive laboratory testing, were not associated with improved outcomes. These findings are not surprising as laboratory schedules tend to be protocol driven at some centers, whereas at other sites NICU clinicians may individualize laboratory testing based on individual clinical indications. The Neonatal Research Network hypothermia trial left surveillance for organ dysfunction at the discretion of treating physicians and described as “per routine clinical care.”39 Neuroimaging costs such as brain MRI were not included in this study given that this is routinely performed after the rewarming period at most centers. Furthermore, brain MRI is a relatively small proportion of overall hospital costs, is not as variable compared with other categories of costs,2 and is performed mainly for prognostic rather than interventional purposes. Additionally, our cost analysis was limited to hospital charges coded by PHIS, therefore, this study did not consider costs of aEEG, hours of monitoring, nor account for professional charges that may not be captured in PHIS. We also acknowledge that there may be differences between centers not accounted in this study that can affect patient outcomes.This study has several limitations which include being a registry study and observational in nature, and therefore has bias inherent to retrospective analyses (despite prospectively collected data). We evaluated costs during the first 4 days of age to focus on the period of NNCC during TH and rewarming and to reduce the variability in costs that are related to differences in length of hospital stay. We were interested in the most acute period of care with likely the highest variability in practice to assess whether investment in neurocritical care during this period affects survival and neurodevelopmental outcomes. This approach is consistent with a prior study where illness severity during the first 4 days of age, not just during the initial hypoxic event, was shown to be a predictor of adverse outcomes in neonates with HIE treated with TH.Higher resource utilization as measured by hospitalization costs during the first 4 days of age for neonates with HIE treated with TH was not associated with neurodevelopmental outcomes. Infants cared for in centers ranked in the highest tercile for EEG costs had lower odds of death or NDI, whereas higher laboratory costs were not associated with improved outcomes. These findings may serve as the first steps toward identifying aspects of NNCC which relate to neurodevelopmental outcomes and guide future care in this patient population.


De-implementation of low-value care can increase health care sustainability. We evaluated the reporting of direct costs of de-implementation and subsequent change (increase or decrease) in health care costs in randomized trials of de-implementation research.We searched MEDLINE and Scopus databases without any language restrictions up to May 2021. We conducted study screening and data extraction independently and in duplicate. We extracted information related to study characteristics, types and characteristics of interventions, de-implementation costs, and impacts on health care costs. We assessed risk of bias using a modified Cochrane risk-of-bias tool.We screened 10,733 articles, with 227 studies meeting the inclusion criteria, of which 50 included information on direct cost of de-implementation or impact of de-implementation on health care costs. Studies were mostly conducted in North America (36%) or Europe (32%) and in the primary care context (70%). The most common practice of interest was reduction in the use of antibiotics or other medications (74%). Most studies used education strategies  (64%). Studies used either a single strategy (52%) or were multifaceted (48%). Of the 227 eligible studies, 18 (8%) reported on direct costs of the used de-implementation strategy; of which, 13 reported total costs, and 12 reported per unit costs (7 reported both). The costs of de-implementation strategies varied considerably. Of the 227 eligible studies, 43 (19%) reported on impact of de-implementation on health care costs. Health care costs decreased in 27 studies (63%), increased in 2 (5%), and were unchanged in 14 (33%).De-implementation randomized controlled trials typically did not report direct costs of the de-implementation strategies (92%) or the impacts of de-implementation on health care costs (81%). Lack of cost information may limit the value of de-implementation trials to decision-makers.https://osf.io/ueq32.OSF (Open Science Framework): The online version contains supplementary material available at 10.1186/s13012-023-01290-3. Contributions to the literatureThe need for economic information has been identified in the field of implementation science. The same should be expected for studies reporting de-implementation strategies.The costs and impact on health care costs of de-implementation strategies are currently seldom reported in randomized trials on de-implementation. Even when they are reported, the information is incomplete and scarce.To improve health care quality and effective resource use, de-implementation strategies need to measure clinically relevant outcomes, and the trials also need to report intervention costs and impact on health care costs.Efficient use of health resources benefits both individuals and society — and one way to increase efficiency is to abandon obsolete and ineffective health interventions , 2. De-iDifferent terms are used from these withdrawn actions, like de-implementation and disinvestment . Public Economic evaluation has been pointed out to be crucial part of implementation research –16, and The above considerations are equally valid when de-implementation is concerned  as de-imSince the aim of implementation and de-implementation is similar — to improve the quality of health care and effective use of resources — de-implementation strategies need to measure clinically relevant outcomes but also to analyze whether a strategy leads to a change in health care costs. The potential savings in health care costs as well as the costs of de-implementation strategy itself must be taken into account.The aim of this systematic scoping review was to analyze how de-implementation studies have reported both the costs of de-implementation strategies and the impacts (estimated or measured) of de-implementation on health care costs.We used the PRISMA extension for scoping reviews (PRISMA-ScR)  to guideLiterature searches for these economic analyses are drawn from the registered systematic scoping review and are described in detail elsewhere . We searThe inclusion and exclusion criteria are described previously . In brieTo assess the quality of the included studies, we used a modified Cochrane risk-of-bias tool (RoB2.0) for randomized trials . The proBoth independently and in duplicate, we used standardized forms with detailed instructions in identifying eligible articles (titles and abstract and full-text screening) and in data extraction. Disagreements were resolved through discussion and, if necessary, through consultation of a third investigator.We collected the following data: (1) study characteristics , year, country of origin, sample size), (2) types and characteristics of interventions , (3) characteristics of the practice of interest , (4) outcomes of the study, (5) intervention efficacy, (6) costs of de-implementation , and (7) effect on health care costs . The costs of de-implementation had to be reported in monetary form, and total or per unit cost were specified by the study authors. Data regarding costs of de-implementation and effect on health care costs are reported in this article; other outcomes are reported elsewhere .We summarized the characteristics and details of de-implementation strategies and target population(s) and provided an overview on de-implementation costs. We extracted the costs in the reported currency and converted it into USD in 2021 value to facilitate comparability across all included studies. We changed the currency from EUR to USD, because more studies have used USD. We used a modified Effective Practice and Organisation of Care (EPOC) taxonomy [Finally, we provided an overview of the impact of de-implementation on health care costs. We reported the direction of the effects and cost allocations. We relied on the authors’ conclusion on the significance of the effect. We analyzed possible between-study differences in influence on health care costs. This was reported in various ways .We planned to do subgroup analyses based on (i) health care settings, (ii) target of intervention, (iii) health care financing, and (iv) country income groups. We assumed beforehand that the studies would be heterogeneous so a meta-analysis would not provide any added value.P-values less than 0.05 were considered statistically significant.We used summary statistics  to describe study characteristics. We used nonparametric tests to analyze differences between outcomes of our interest. For statistical analyses, we used IBM® SPSS® version 28.0.1 , and all reported Of the 12,815 articles identified in our search, we evaluated 1022 full-text articles. We included 227 RCTs, of which only 50 (22%) reported any costs or impact on health care costs. Figure n = 18, 36%) and Europe . The majority of the studies  were targeted to one type of professionals, and nine studies (18%) reported several target groups. Around two-thirds of the studies  were conducted in primary care. The trials were aimed at reducing the use of drug treatments , laboratory test , or diagnostic imaging . The studies used 16 different de-implementation strategies. Twenty-six used only one strategy, and twenty-four were multifaceted including two or more strategies. In all studies, the goal was to reduce use of a specific health care intervention. In 14 studies, an additional goal was replacing. The description of the characteristics of the included studies is shown in Table The publication dates of the included articles ranged from 1982 to 2021, where half the articles dated after 2010. Most articles were from North America , 22 studies (44%) had missing data, and 20 (40%) had imbalance in baseline characteristics. Data collectors were blinded in 41 studies (82%) but data analysts in only two studies (4%) . These total costs varied considerably, the median being US $32,300 . Table n = 9), audit and feedback (n = 7), educational meetings for individuals (n = 4), treatment algorithm (n = 3), educational meetings for groups (n = 3), and developing clinical practice guidelines (n = 2). The strategies used in one study included alerts, local consensus process, educational material for patients, and public intervention. Strategy combinations were diverse; many combined different educational strategies together. When using educational material in de-implementation, the median for total costs median was US $118,000 . The total costs seemed higher in studies using educational materials than in studies not using such materials when not using it . For other strategies, the total costs did not significantly differ between studies using vs. from not using each strategy .The 13 studies (26%) that reported total costs used ten different de-implementation strategies. The most common strategies were educational materials , the median for total costs was US $8090 . In studies using two de-implementation strategies , the median for total costs was US $224,000 , and with three or more strategies , the median for total costs was US $43,600 .In studies that used only one de-implementation strategy (n = 8), educational meetings for individuals (n = 6), and for groups (n = 5), audit and feedback (n = 5), and educational materials for patients (n = 2). Alerts, treatment algorithms, public intervention, and developing clinical practice guidelines were each used once. There were no differences in costs between using a given strategy and not using it .Costs per unit were reported in 12 studies (5%). The most common unit was cost per physician, but also costs per health care provider, health care unit, day, and patient were reported Table . In thesOf the articles that reported total costs or costs per unit, 10 out of 18 (56%) offered at least some detailed information on the costs, but only four (22%) reported the exact costs. The most frequently reported types of costs were material costs, payment for trainers, and travel costs. In addition, postage, rent of premises, and loss of working hours were occasionally reported. Cost related to de-implementation intervention planning was rarely brought out. Information on costing methods was not mentioned in the articles. None of the articles separated costs related to the phases of de-implementation.A meta-analysis was not possible due to the heterogeneity of the studies . There were few studies in the pre-specified subgroups, so subgroup analyses were not appropriate.n = 25, 58%). In four studies (9%), the impact was on patients’ own costs, whereas in 14 studies (33%), it was on health care provider’s costs. In 27 (63%) studies, health care costs decreased, whereas in 14 (33%), there was no change, and in two (5%) studies, the costs increased. The impact was targeted to medicine costs , laboratory test costs , total health care utilization costs , diagnostic testing costs , and radiography costs .The impact on health care costs was reported in 43 studies (19%). In most cases, the reports did not specify to whom the impact was targeted  have based their assessments on calculations on differences in costs between intervention and control group. In eight studies (19%), the authors have expanded the intervention group costs changes to large groups or for longer time. In one study [Most of the articles  or had no effect on costs . In six studies, the authors estimated the impact on health care costs. De-implementation influenced laboratory test costs (n = 6), medicine costs (n = 5), diagnostic testing costs (n = 2), radiology costs (n = 2), and total health care expenditure per visit (n = 1). Table The two studies , 31 with n = 2, 1%. In six n = 2, 1%. In sixn = 20, 80%), the de-implementation mainly influenced the costs of medicine and laboratory tests. The change in reported health care cost varied between US $12.6 per patient to US $80.4 million per country. Of these 25 studies, 15 reported the impact in a monetary measure using different currencies  reported a decrease in costs, whereas 11 (44%) reported no change , educational materials , audit and feedback , educational meetings for individuals , treatment algorithm , educational materials for patients , and developing clinical practice guidelines . Two studies used public release of performance data and patient-mediated interventions. The strategies used in one study included financial incentives for health care workers, local consensus process, local opinion leaders, managerial supervision, and routine patients-reported outcome measures.The 43 studies that reported impact on health care costs used 15 different de-implementation strategies. The most common strategies were educational meetings for groups , while unit costs and impact on health care costs were reported in five (10%) articles Table . The artEven though de-implementation is often justified by emphasizing control of health care costs , 7, 32, For economic evaluation, information on resource use, costs, time horizons, health outcomes, or the consequences of interventions are necessary . IncomplTo improve the utilization of de-implementation research, economic evaluation should be planned along with the research. Subsequently, the studies should report precise monetary costs of de-implementation strategies and their impact on health care costs. When reporting costs, general considerations should be taken into account: (i) give detailed and reasoned values, (ii) separate included costs, (iii) provide the time horizon when the costs are applicable, and (iv) break down the planning and acting phase costs of the de-implementation process.Our highly sensitive literature search used a wide variety of de-implementation terms. However, due to heterogeneous indexing of de-implementation studies, it is possible that we may have missed relevant articles.A strength of our article is that we searched for cost information and de-implementation impact information from the full text of articles, which noticeably increased the number of included articles — as the impact on health care costs tended to be reported in the full text, not in the abstract.We restricted our study to RCTs, which may be seen as a limitation. Since many de-implementation projects have likely not included randomized control groups, we missed economic information from these studies. However, the efficacy of interventions should be studied in RCT settings, and thus, we believe that our decision to exclude other study designs is justified.This review was made alongside with another review, which may have restricted the number of included studies. We excluded studies where one medical practice was used to de-implement another, because these often focus on implementation not on de-implementation. We focused on de-implementation of low-value care, and therefore, excluded articles were the reason for de-implementation which was cutting resource use. Both these restrictions may have excluded some articles where cost information could have been given. However, we believe that our careful selection of articles in the full-text phase has prevented this. Our perspective was to find out how costs are brought out in de-implementation studies, so the search was made on that view. It could be a limitation, and some studies with costs may have been missed. To avoid this, we searched also the references of included studies to find other articles on same studies. Using the approach that Vale has used, it may lead to a different result.A vast majority of de-implementation trials have failed to report any intervention costs or impacts on health care costs. In studies that do include cost information, typically only nonnumerical information on economics impacts is reported, and direct costs of de-implementation strategies are excluded. To advance the field, researchers should consider economic aspects and include health economists when planning research. De-implementation interventions are often complex and resource intensive, and cost information is essential for effective health policymaking.Additional file 1. PRISMA-ScR checklist.Additional file 2. Search strategy.Additional file 3. RoB tool.Additional file 4. List of included studies.Additional file 5. Characteristics of included studies.


The history of the Romanov breed goes back more than a century, with the first mention being made in 1802. This breed has unique qualities, such as a good resistance to harsh environments, high litter size, polyestrousity, early maturation and excellent fur coat qualities. In this work, we highlighted several genetic markers involved in these characteristics. The high ovulation rate of the Romanov breed is probably linked to the polymorphism recorded in two genes located on different chromosomes. The breed’s adaptation to the harsh environment of its breeding area in the Russian Federation could be associated to a high level of heterozygosity. In addition, the dilemma of its obscure origin was also addressed by comparison with other Russian breeds of different productive orientations.AHB than BHB alleles. There are 3–4 genotypes at the transferrin locus whereas in other breeds 6–11 genotypes have been found. At the albumin locus, the majority of the identified genotypes were heterozygotes, unlike in the other breeds studied. In the prealbumin locus, the Romanov breed was the only one where all the genotypes were heterozygous. We speculate that polymorphism at two loci (BMP-15 and BMPR1B) could effect on the high ovulation rates of Romanov sheep. Based on different genetic markers, the prevalence of heterozygotes in the Romanov sheep could determine their higher viability. A cluster analysis showed the close proximity of 12 populations of the Romanov breed, as the breeding stock come from the Yaroslavl region.The Romanov breed was evaluated using immunological and genetic markers. The seven blood group systems were characterized with a greater accuracy than in previous works on sheep in the Russian Federation, and were compared to eight ruminant species. Unlike other breeds, Romanov sheep shows a higher frequency of  In the central zone and in the European north of the Russian Federation, the traditional Romanov sheep is one of the important breeds for meat production and fur coats. The Romanov breed is one of the 200 most famous sheep breeds in the world. Animals of this breed are characterized by exceptional fertility. Under normal conditions of feeding and rearing, 100 ewes give birth to 260–300 lambs per year, and the selection of the most prolific parental animals allows to breed this character , HFecX 544 (C-T), IFecX [579 (T-A)], LFecX [635 (G-A)] and BFecX [773 (G-T)], primers defined in , HFecX [544 (C-T)], IFecX [579 (T-A)], LFecX [635 (G-A)], BFecX [773 (G-T)]) ) . Among tmorphism . The freentified . As for GFecX and BFec, respectively, without double heterozygotes. It means than more than a half of this ewe population carry a mutation enhancing the ovulation rate. Moreover, as carriers of both mutations certainly exist, they could have, themselves, a higher ovulation rate than those with either mutation separately [BFec in the BMPR1B gene. Later, this mutation was also found in coarse wool sheep breeds  in China [BMPR1B (BFec) gene in the studied Tomanov ewes is possibly due to Asian roots of origin. Judging by the works of academician N.I. Vavilov, the flow of animals and consequently genes to the Russian Federation came from China, through Mongolia and Kazakhstan [GFecX mutation, it has been found in breeds of very distant countries: Lleyn in North Wales and Small-tailed Han in China, suggesting independent mutation events [If the 2 loci are considered together, 13 and 14 heterozygotes were found for parately . At presparately , a carriin China ,25,26. Tzakhstan . As for n events . This siAHB than the BHB allele was found, in contrast to most sheep breeds in the world, where the old BHB allele is predominant (HB) locus. They have a prevalence of BHB over the AHB allele, although all the studied breeds have been bred for a long time in rather specific environmental conditions. It should be noted that the HBA allele is a derivative of BHB, associated with a deletion, i.e., loss of a DNA segment of 40 kb. The high occurrence of the BHB allele in most of the studied sheep suggests that most breeds were created from populations that carried the “old” BHB allele [In the Romanov breed, a higher frequency of dominant . The coaB allele . A/AHB homozygotes or A/BHB heterozygotes are more resistant to the conditions of high humidity in Central Russia. For at least one century, the Romanov breed has been reared in a closed regime, without interbreeding with other breeds. A polymorphism at the hemoglobin locus would create opportunities for an easier oxygen attachment by the A variant of the protein, which allows this sheep to survive for a long time in unusual environmental conditions. This situation suggests that gene frequencies for hemoglobin types can serve as a population indicator of extreme conditions caused by different degrees of hypoxia. Establishing the adaptive significance of the studied traits at different levels of biological organization could make it possible to identify a certain role of biochemical polymorphism in the ecological and genetic differentiation of animals [We hypothesize that sheep being  animals .The function of transferrin is to transport iron ions to reticulocytes, in which hemoglobin biosynthesis takes place. It is the most polymorphic blood protein locus in sheep. The transferrin locus polymorphism was analyzed in 10 sheep breeds of Russia , Azerbaijan  and Moldova (Tsigay and Karakul) of different productive orientations . ATF, BTF, CTF, DTF, ETF, PTF, ITF—the combination of which theoretically gives twenty-eight phenotypes. Specifically, 20 transferrin phenotypes have been identified: A/ATF, A/BTF, A/CTF, A/DTF, A/ETF, A/ITF, B/BTF, B/CTF, B/DTF, B/ETF, B/ITF, C/CTF, C/DTF, C/ETF, C/ITF, D/DTF, D/ETF, D/ITF, D/PTF, E/PTF. Phenotypes containing rare alleles have not been detected: A/PTF, B/PTF, C/PTF, E/ITF, P/ITF. There were no genotypes homozygous for rare alleles . This phenomenon was observed in all three studied populations of Romanov sheep. The most common alleles were ATF, BTF, CTF and DTF, rarely ETF, ITF and PTF. The CTF allele had the highest frequency among all the studied breeds . In the Romanov and coarse wool breeds, the individuals shared a low frequency of the ATF allele. In fine wool and semi-fine wool sheep, the number of genotypes varied between eight and eleven, thus being higher than in the Romanov sheep, with five to six genotypes. In coarse wool sheep, the range is between 5 and 13 genotypes. Seven transferrin alleles were identified—The studied sheep breeds were characterized by a high level of heterozygosity. The Caucasian breed shows the lowest value with 0.48, while the highest one was observed in the third population of the Romanov breed with 0.93. In the populations with a heterozygosity over 0.73, two showed a deviation from genetic equilibrium (the third population of Romanov and Karakul), but not in North-Caucasian meat-wool. In the Caucasian breed, the deviation was mainly due to the absence of BC heterozygotes.Albumins are synthesized in the liver and are simple proteins containing up to 600 amino acid residues. They are the lowest molecular weight blood proteins, with values of about 60,000–66,000 D. Their function is to maintain the colloid osmotic pressure of the plasma, the constancy of the concentration of hydrogen ions, and also the transport of various substances. Normally, albumins account for 35–55% of the total amount of blood plasma proteins  and can p < 0.05) with 0.90 compared to 0.758, respectively. The populations showing a significant deviation from the Hardy-Weinberg genetic equilibrium showed heterozygosity values higher or equal to 0.875. Among all the studied breeds, this relates to the three populations of Romanov, the North Caucasian meat-wool and the Volgogradsk breeds.The comparison of genetic richness in sheep with different production characters showed that in the coarse wool breeds, including Romanov, the mean number of genotypes was higher than 5.5 in the semi-fine and fine wool breeds, but lower than this value in the semi-fine and fine wool sheep . The VolA allele, most likely due to a lower viability of the carriers of these genotypes. There is a great demand for heterozygous genotypes in sheep breeds, regardless of the productive specialization and place of breeding.In semi-fine wool  and fine wool  sheep, the albumin locus showed an absence or rare occurrence of genotypes with the ALBIn addition to the Romanov breed previously studied , the polF/FPRE, F/SPRE, F/OPRE, S/SPRE and S/OPRE. The level of heterozygosity in the studied breeds was above or equal to 0.8 and even reached 1 in each of the 3 populations of Romanov. It should be noted that in all breeds, all phenotypes of the prealbumin protein were found, with the exception of the O/OPRE variant, which is possibly associated with the non-viability of the offspring. In most of the studied breeds, there was a non-significant deviation from genetic equilibrium in this locus, with the exception of the Caucasian breed. The Romanov breed presented the highest deviation of all the studied breeds.In the prealbumin locus, five out of six putative genotypes were identified. The majority of the possible genotypes of this protein were detected among the 10 breeds: The comparison of the richness in alleles and genotypes between breeds of different characteristics shows an occurrence of five genotypes and three alleles in fine wool, semi-fine wool and coarse wool breeds. In contrast, there were only three genotypes in the Romanov sheep. As in the albumin locus, the prealbumin locus of the Romanov breed is characterized by an under-representation in homozygous genotypes compared to the other studied breeds, even in the coarse wool group; this may be indicative of a selective advantage of heterozygotic animals.t-test, p = 0.0076 and 0.031 for heterozygosity and allelic richness, respectively). It can be concluded that the Romanov belongs to the breeds presenting the highest genetic variability among the northwestern cluster of coarse wool breeds.An analysis of 20 microsatellite loci on 26 sheep breeds of Eurasia revealed that the Romanov breed was close to the Russian Kulunda settled in the Altai region, and loosely related to 2 Polish breeds (Swiniarka and Wrzosowska) and 4 Scandinavian ones  . Using tInterestingly, Ozerov et al.  found thE); level of observed heterozygosity (HO); allelic richness, i.e., number of alleles at the locus (AR) averaged over the minimum sample size (n = 38); mean number of alleles per locus (AM); coefficient of inbreeding (F)  .O) was found in sheep from farm No. 2 of the Voronezh region , and the lowest level was found among sheep from farm No. 10 of the Yaroslavl region . In the rest of the farms, the level of heterozygosity was intermediate. The maximum level of allelic richness (AR) was observed among sheep from the farm of the Republic of Bashkortostan (No. 1: 4.61); the minimum values were detected among sheep from the Smolensk region (No. 4: 2.56). The highest number of alleles per locus (AM) was observed in sheep from the Republic of Bashkortostan (No. 1) and farm No. 3 from the Voronezh region (4.71), and the minimum among sheep of farm No. 4 of the Smolensk region. The coefficients of inbreeding (f) did not significantly deviate from zero in any of the studied populations.The analysis showed that the highest level of observed and expected heterozygosity  and BFec (BMPR1B gene) were identified for the first time in the Romanov breed. Regarding the hemoglobin locus, the previous results obtained for the Romanov breed in the paper of 2015 [A high ovulation rate is one of the important features of Romanov sheep. A description of the polymorphism of the The history of Romanov sheep is poorly understood; there are several hypotheses, although the first mentions date back to the 19th century . It is tATF of the transferrin locus and a more frequent allele OPRE in the prealbumin locus. Regarding the originality of the Romanov breed, the pattern of alleles within the different blood systems seems to be unconnected to the three Ukrainian breeds, but this result is preliminary given the lack of homogeneity in the accuracy of analyses. The prevalence of the b AHB allele in the hemoglobin locus is unique among the coarse, semi-fine and fine wool analyzed breeds, for which the allele BHB prevails. In the transferrin locus, the CTF allele is dominant in all the breeds investigated, but the Romanov breed shows the highest frequency. In the albumin locus, the Romanov sheep differed from the others due to its higher frequency of allele AALB and lower frequency of BALB. In the prealbumin locus, the Romanov is the only breed without carriers of homozygous animals FF and SS. Taken together, these features suggest that the Romanov sheep experienced a genetic isolation from other breeds for a long period. Unexpectedly, the discovery of the BFec mutant (Booroola) in the BMPR1B gene in the Romanov sheep raises the question of its appearance, probably dating back several centuries. Having arisen on the Hindustan Peninsula, the mutation, by chance, ends up in Australia, where a new breed was being formed [The connection between the Romanov and coarse wool breeds is supported by a weak piece of evidence: a common low frequency in the allele g formed ,23. Lateg formed ,24,25,34Regarding the adaptation of the Romanov sheep to the harsh conditions in the central regions of the Russian Federation, several traits of its fur have been already mentioned. Our results indicated the high degree of heterozygosity of this breed for several loci. First of all, only heterozygotes were found in the prealbumin locus, and this was nearly also the case for the albumin locus in two populations among the three investigated, in contrast to other breeds. Moreover, the data based on microsatellites emphasized the genetic variability of the Romanov sheep among the northwestern cluster of coarse wool breeds. The reason for interest in the heterozygosity recorded in the populations of Romanov sheep is that it could lead to a higher range of activity of the proteins coded by the different alleles. However, the resistance of this breed could also be due to advantageous variants, as hemoglobin is encoded by the A allele.From a genetic point of view, the following measures are currently in place or will be adopted to maintain the Romanov breed’s genetic variability in several farms within the Yaroslav region: rotation of breeding sheep in order to avoid inbreeding; mandatory testing of the breeding core for genetic markers; creation of a sperm bank from elite rams and embryos from ewes.


Flavivirus genus (family Flaviviridae). QBV has been specifically documented in Vietnam and China, with reports limited to several mosquito species.Mosquitoes carry a variety of viruses that can cause disease in humans, animals and livestock. Surveys for viruses carried by wild mosquitoes can significantly contribute to surveillance efforts and early detection systems. In addition to mosquito-borne viruses, mosquitoes harbor many insect-specific viruses (ISVs). Quang Binh virus (QBV) is one such example, categorized as an ISV within the Aedes albopictus) and BHK-21 (baby hamster kidney) cell lines. Positive cultures were identified by reverse transcription-polymerase chain reaction (RT‒PCR) with taxon- or species-specific primers. Next-generation sequencing was employed to sequence the complete genomes of the identified positive samples. Subsequently, phylogenetic, gene homology, molecular evolutionary and genetic variation analyses were conducted.The homogenate obtained from female mosquitoes was cultured on C6/36  RNA sequences. Phylogenetic analysis indicated that the three Guizhou isolates, along with the prototype isolate from Vietnam, formed distinct branches. These branches were primarily closely related to other QBV isolates reported in China. Comparative analysis revealed a high degree of nucleotide and amino acid homology between the Guizhou isolates and both Vietnamese and other indigenous Chinese isolates. Additionally, nonsynonymous single-nucleotide variants (SNVs) were observed in these strains compared to the QBV prototype strain.In 2021, a total of 32,177 adult female mosquitoes were collected from 15 counties in Guizhou Province, China. The predominant mosquito species identified were Cx. tritaeniorhynchus mosquitoes in Guizhou Province, China. Phylogenetic tree analysis showed that the three Guizhou isolates were most closely related to the QBV genes found in China. In addition, the study of the genetic characteristics and variation of this virus provided a deeper understanding of QBV and enriched the baseline data of these insect-specific flaviviruses (ISFVs).This study represents the first report of QBV presences in The online version contains supplementary material available at 10.1186/s13071-023-05938-3. Aedes spp. and Culex spp. are particularly important in transmission of flaviviruses (family Flaviviridae), alphaviruses (family Togaviridae), bunyaviruses  and seadornaviruses (family Reoviridae). Flaviviruses have caused numerous emerging and re-emerging arboviral diseases [Flavivirus genus, except for Japanese encephalitis virus and yellow fever virus, presenting a significant health challenge.Mosquitoes serve as significant vectors for various arboviruses, causing a significant burden on human health globally . These adiseases , 6. Notadiseases , 8. Simidiseases . Additiodiseases , 11. DesAedes aegypti cells and classified as cell-fusing agent virus, which belongs to the family Flaviviridae [Aedes macintoshi larvae and pupae in 1999 [Culex flavivirus [Aedes flavivirus [The flavivirus can be categorized based on their transmission vectors into three groups: tick-borne flaviviruses, mosquito-borne flaviviruses and flaviviruses with no known vectors , 13. Addiviridae . Subsequ in 1999 . Besidesavivirus  and Aedeavivirus . Since 1avivirus .Guizhou Province, with its diverse natural environment, has been a hotspot for various mosquito-borne diseases and viruses, including Japanese encephalitis and Zika virus –23. HoweFlavivirus (family Flaviviridae) was initially isolated from Culex tritaeniorhynchus in Quang Binh City, Vietnam, in 2002 [Quang Binh virus (QBV) in the genus  in 2002 . Subsequ in 2002 , 26, 27.2  and a portable electroaspirator. Adult mosquitoes were frozen at − 20 °C for 30 min and placed on ice for morphological identification using a dichotomus key [Mosquitoes were collected from July to September 2021 from surveillance sites in 15 counties and districts in Guizhou Province, China   and inoculated into 12-well plates of C6/36  and BHK-21 (baby hamster kidney) cells for 1 h with 100 μl filtrates, with two wells for each sample. Next, 1 ml of media containing 2% serum was added, and the incubation was continued for 6–7 days. Two wells in each plate were used as negative controls. C6/36 cells were maintained in RPMI 1640 medium containing 2% fetal bovine serum  at 28 °C with 5% CO2, and BHK-21 cells were maintained in minimal essential medium  containing 2% FBS at 37 °C with 5% CO2. All media were supplemented with 1% penicillin-streptomycin . Cells were scored daily for cytopathic effects (CPEs), and cultures were harvested from the plates when > 75% of the monolayer was affected by CPE. Positive cultures were processed three times to obtain pure cultures. If no cytopathic effect was observed after three passages, the sample was considered negative.The virus was successfully isolated using the Vero cell culture method . After thttps://sg.idtdna.com/PrimerQuest/Home/Index). PCR was performed using a 2 × SuperNova PCR Mix  PCR system , which contained 25 μl of 2 × SuperNova PCR Mix, 2.5 μl of each primer (10 μM), 18 μl of sterile water and 2.0 μl of cDNA sample. A thermal cycling procedure  was performed at 98 °C for 3 min, followed by 30 cycles at 98 °C for 15 s, 55 °C for 15 s and 72 °C for 30 s, with a final extension step of 5 min at 72 °C. The amplified products were analyzed by agarose gel electrophoresis and then purified and sequenced in both directions by Sangon Biotech . BLAST alignment was performed on the above sequences (http://www.ncbi.nlm.nih.gov/BLAST).Total RNA was extracted from cell-positive cultures using the QIAamp Viral RNA Kit , and total RNA was reverse transcribed using the cDNA Reverse Transcription Kit . For the detection of flaviviruses and alphaviruses, polymerase chain reaction was performed using previously published methods and primers , 32. Twog for 30 min at 4 °C  to remove the debris. Twenty microliters of virus suspension was placed with a pipette gun and dropped on a copper grid with carbon film for 3–5 min, and then filter paper was used to absorb the excess liquid. Then, 2% phosphotungstic acid was dropped on the copper grid to stain for 1–2 min, filter paper was used to absorb excess liquid, and the grid was dried at room temperature. The copper grids were observed under transmission electron microscopy and photographed.The positive virus culture supernatants from the cells were collected and centrifuged at 20,000 × g for 2 min to remove cells. Virus supernatants were treated with 200 U Benzonase (Millipore), Tubro DNase I (Thermo Fisher Scientific) and 0.1 mg/ml RNase A (Sangon Biotech) followed by heat inactivation of DNases at 65 °C for 10 min. Viral RNA was then extracted using the Qiagen MinElute Virus Spin Kit according to the manufacturer's instructions. RNA was quantified using an Equalbit RNA HS Assay Kit . The RNA virome library was constructed using a sequence independent amplification method [The virus supernatant was centrifuged at 12,000×n method . The lib–5. According to the blast score, the virus species with the highest identity was selected as the final annotation for a query contig. First, the reference genome sequence was downloaded: Quang Binh virus (MH827524). Then, the contigs annotated to the same virus were mapped to the corresponding reference genome using MegaBLAST with “-dust no -word size 18.” The mapped contigs were then manually assembled using the Seqman application in the DNASTAR software package. All reads were remapped to the annotated viral contigs using BWA [Raw data were processed using Fast  by filtesing BWA  for quan5′ RACE was performed using the HiScript-TS 5′/3′ RACE kit  following the manufacturer's instructions. RNA was extracted from positive virus isolates, and the quality of RNA was assessed by gel electrophoresis or bioanalyzer. The primers used for 5′ RACE were as follows and designed based on known sequence information: TTT GTT TTC CCC TCG TAG ACC TGC ACG C (5′ specific). The PCR products were purified and cloned. Sanger sequencing was then performed to determine the unknown end of the target sequence.Culex flavivirus, Aedes flavivirus, yellow fever virus, Japanese encephalitis virus, West Nile virus and dengue virus types 1–4 (DENV 1–4),  and the DNAStar (Lasergene) program package (https://www.dnastar.com/). To generate a heatmap, TBtools [https://github.com/CJ-Chen/TBtools/releases). A phylogenetic tree was constructed with MEGA11 [http://tree.bio.ed.ac.uk/software/figtree/).Multiple sequence alignments were performed with the gene sequences of three Guizhou viruses and relevant viral sequences using MAFFT version 7.490 . The seq TBtools  was usedh MEGA11  using thGene annotation of three Guizhou virus strains was referenced to QBV (NC_012671). Briefly, we first downloaded two major protein sequences from QBV genomes as input and then used Prokka software  to acquiFor sequence variant detection, the complete genomes of each Guizhou virus strain were aligned to QBV using ClustalW , and theCulex flavivirus (AB262759.2) and Quang Binh virus (FJ644291) have been downloaded from NCBI. We manually searched for five match patterns that have been reported in insect flaviviruses . Furthermore, RNA secondary structure prediction was performed 150 bp downstream of the matched site by using RNAfold software [With reference to the published literature, the reference genomes of Hanko virus (JQ268258), software , and onlAnopheles (An.), Culex (Cx.), Aedes (Ae.) and Armigeres (Ar.). Among the collected mosquitoes, Culex accounted for 53.5% , followed by Armigeres at 34.1% , Anopheles at 11.7%  and Aedes at 0.8% . The dominant species was Cx. tritaeniorhynchus, comprising 48.7%  of the collected mosquitoes, followed by Armigeres subalbatus  and Anopheles sinensis .A total of 32,177 mosquitoes were collected from 15 counties of Guizhou Province, China. These mosquitoes were sampled from various locations, such as pig pens, cattle pens, trees, houses and other places. Among the collection sites, the largest portion of mosquitoes was obtained from cattle pens, accounting for 47.8%  of the total, followed by pig pens at 41.9% . These collected mosquitoes belonged to six species from four genera and two subfamilies, namely In this study, mosquito samples were divided into 200 pools based on the location, time of collection and mosquito species, and virus isolation was performed on each pool by inoculating onto C6/36 and BHK-21 cells.Flavivirus, family Flaviviridae). Of these, three virus isolates were from the Cx. tritaeniorhynchus pools, and they were collected from three areas  exhibited the characteristic organization commonly found in flavivirus genomes. The complete genome sequencing of GZ21m081 revealed a total genome size of 10,831 nt, consisting of a 98-nt 5′-end noncoding region, 10,080-nt coding region and 653-nt 3′-end noncoding region. Similarly, the total genome size of GZ21m120 was 10,787 nt, with a 98-nt 5′-end noncoding region, 10,080-nt coding region and 609-nt 3′-end noncoding region. For GZ21m167, the total genome size was 10,678 nt, but the 5′-end noncoding region was not amplified. A 632-nt fragment was amplified using 5′ RACE, and a 10,829-nt viral genome was obtained via sequence splicing. The sequence consisted of a 117-nt 5′-end noncoding region, a 10,080-nt coding region and a 632-nt 3′-end noncoding region. BLAST analysis confirmed the similarity of the complete genome sequences in this study to other QBV isolates and references. The three genome sequences have been registered in the NCBI GenBank with the following sequence numbers: OQ139646, OQ139647 and OQ139648.Flaviviridae family: mosquito-borne flaviviruses, tick-borne flaviviruses, no-known vector flaviviruses and ISFVs. The analysis of the sequences  from Guizhou revealed that they belonged to the insect-specific flaviviruses and were grouped in the QBV clade. Interestingly, QBV exhibited two distinct branches in the clustering analysis. One branch comprised mainland Chinese strains from Shanghai, Liaoning, Jiangsu and Guangdong, while the other branch contained the first reported QBV strain from Vietnam (VN180) and a strain from Guangdong, China (MH827523). When compared to other members of Flavivirus genus, all Guizhou virus isolates formed a distinct group alongside QBV strains from China. The phylogenetic tree of the complete genome sequence  and deduced amino acids . Heatmap analysis revealed that the three Guizhou strains were highly homologous to QBV at the nucleotide and amino acid levels Fig. . CompareCircular maps, BLAST analysis and nonsynonymous SNV were used to illustrate three Guizhou virus genomes Fig. . NucleicCompared to the reference sequences , the three Guizhou genomes exhibited 1050, 1049 and 1058 SNV sites, respectively. Among these SNVs, there were 104, 106 and 105 nonsynonymous SNVs, respectively  followed by a predicted occurrence in the NS2B region. However, HANKV had a different conserved sequence (G_GAU_UUU) in the NS2A-NS2B region. The frameshift site started at position 3333 (HANKV) to 3419 GZ21m167). Additionally, a predicted RNA stem-loop structure was identified in the NS2B region  [The squitoes . Advancespecies) , 49, 50.Cx. tritaeniorhynchus, accounting for 48.7% of the collected samples, followed by Ar. subalbatus mosquitoes at 34.1%. The mosquito samples were primarily collected from cattle and pig pens. Culex tritaeniorhynchus vector, being the most common mosquito species in the province, plays a crucial role in the virus transmission, particularly as the principal vector of Japanese encephalitis [During our study, we found the predominant mosquito species in Guizhou province was phalitis . TherefoCx. tritaeniorhynchus in the three regions of Guizhou province  within the QBV group. The analysis revealed that these three Guizhou virus sequences clustered with ISFVs QBV found in Chinese provinces Fig. . Two maiFurthermore, the complete sequencing of the three Guizhou isolates allowed for a better understanding of their genetic relationships. The different regions of the sequences exhibited > 84% nucleotide similarity to the corresponding regions of the prototypical QBV isolate Fig. . A speciAdditionally, we conducted SNV analysis of the ORFs of the three Guizhou virus genomes and compared them with the corresponding regions of prototype QBV isolates. The analysis revealed that there were more synonymous mutations than nonsynonymous mutations, and the mutation sites and types were largely consistent among the three Guizhou virus genomes Table , Fig. 6.fifo, in the NS2A-NS2B region [Ribosomal frameshifting, specifically − 1 ribosomal frameshifting, is a well-defined process where ribosomes shift by one nucleotide and translate in a new reading frame . VirusesB region , 61. In B region , althougMany aspects of the interaction among ISFVs, hosts, vectors and humans remain unclear , 64. SurCx. tritaeniorhynchus in Guizhou Province. Phylogenetic analysis revealed that the three Guizhou isolates were most closely related to the QBV strains found in China. Furthermore, our comprehensive analysis of the complete genome and nucleotide variation in different genomic regions offers valuable insights into QBV evolution. This study significantly expands the baseline data of QBV genomes and provides a valuable resource for future research on molecular epidemiology, evolutionary studies and the development of molecular assays.Our study on wild mosquitoes carrying QBV demonstrates the One Health approach. It is the first report of QBV in Additional file 1: Table. S1. Background information on the Quang Binh virus analyzed in this study.Additional file 2: Table. S2. Data of the circular diagram on nucleic acid similarity and nonsynonymous SNV analysis in this study.Additional file 3: Table. S3. The results of nonsynonymous and synonymous SNV analysis in this study.