Vitivirus have been associated with the grapevine rugose wood disease. Based on new and previously reported GVG isolates, primers and probes were developed for real-time RT-PCR. The developed assay successfully detected the virus in infected plants during dormancy and the growing season. A field study of 4327 grapevines from Croatian continental and coastal wine-growing regions confirmed the presence of GVG in 456 (~10.5%) grapevines from three collection plantations and 77 commercial vineyards, with infection rates ranging from 2% to 100%. Interestingly, the virus was confirmed only in vines considered to be Croatian autochthonous cultivars, but not in introduced cultivars. A 564-nucleotide long portion of the coat protein gene from previously known and newly characterized GVG isolates had nucleotide and amino acid identities ranging from 89% to 100% and from 96.8% to 100%, respectively. Phylogenetic analysis revealed five distinct groups, with isolates originating from the same site being close to each other, indicating possible local infection. The information presented in this manuscript sets the stage for future studies to better understand the ecology and epidemiology of GVG and the possible need for inclusion in certification schemes.Grapevine virus G (GVG) is a recently discovered vitivirus infecting grapevines. Historically, viruses in the genus  Vitivirus (family Betaflexiviridae) includes most grapevine-infecting viruses. According to the current taxonomy of the International Committee on Taxonomy of Viruses , the genus Vitivirus consists of 15 viruses, nine of which are grapevine-infecting: grapevine virus A (GVA), grapevine virus B (GVB), grapevine virus D (GVD), grapevine virus E (GVE), grapevine virus F (GVF), grapevine virus G (GVG), grapevine virus H (GVH), grapevine virus I (GVI) and grapevine virus J (GVJ) [The genus  J (GVJ) ,7,8,9. I J (GVJ) ,11,12,13 J (GVJ) .Trichovirus [Vitivirus. ORF1 encodes a polyprotein, ORF2 encodes a 154 aa long protein, ORF3 comprises the viral movement protein domain, ORF4 encodes the Tricho CP domain of 201 aa, while ORF5 contains the viral nucleic acid binding domain [The best studied ones are also the first grapevine-infecting vitiviruses discovered: GVA and GVB, which were segregated from the genus chovirus . They archovirus ,17. Infechovirus . On the g domain . After tg domain . Later, Although HTS is more sensitive compared to the other detection methods  and allows for novel virus detection and screening of a much larger set of samples (pool) in a time and space frame, PCR-based detection methods are still better suited for large-scale viral testing, thus providing much higher detection sensitivity and specificity compared to other diagnostic methods, such as biological indexing and enzyme-linked immunosorbent assays—ELISA . TherefoAfter alignment of all 11 available GVG sequences from the GenBank database and the sequences of the two newly characterized GVG isolates via HTS, VVL-150 and VM-160 , the CP A validation test of real-time RT-PCR performed on a GVG-positive grapevine from the Grapevine Virus Collection and processed in three replicates of 10-fold serial dilutions, showed that the assay was able to detect virus down to a dilution of 1:100,000. Analysis of the corresponding standard curves for real-time RT-PCR using the mixture of developed primers and probes indicated that the efficiency was 106.869% with a coefficient determination of 0.994 .To determine the potential problematic period for sampling, and consequently to avoid false-negative results during large-scale screening of GVG incidence, detection in different periods of vegetation and dormancy was performed during 2019. As a result, GVG was successfully detected by real-time RT-PCR, with the Cq threshold set at 35, from all five grapevine accessions  during all 16 sampling dates covering the dormant and active growth periods. The only exception was accession VVL-122, which was not tested on March 1, because the sample was spoiled under inadequate storage conditions. Cq values for GVG and 18S rRNA determined for different sampling dates and between different grapevine accessions, as well as their mean values and standard deviations, are provided in The Rq values obtained on April 4, the first sampling date at the beginning of active growing, were significantly lower compared to those obtained between 1 June and 26 November, but without significant differences when compared to other sampling dates . In addition, Rq values near the end of the active growing season (21 September and 23 October) were significantly higher than those obtained between 1 March and 20 August and 26 November and 15 December, except for the values on 1 September and 10 October. The highest Rq values were recorded on 13 November, the end of the active growing season, and were significantly higher than all sampling dates except 23 October. Finally, during dormancy, mid-November to March, there were no significant differences between the mean Rq values. A comparison of mean Rq values among the different grapevine accessions showed no significant differences .In four collection plantations in Zagreb  with 878 vines tested, 30 vines of autochthonous cultivars showed to be GVG-positive (3.4%), of which 22 were found in the Grapevine Virus Collection and 8 in the second National Collection in “Jazbina”. The presence of virus was not confirmed in the first National Collection in “Jazbina”, along with the Grapevine and Rootstock Collection. In the Split Collection, the only one located in the coastal region, out of 105 analyzed samples, 5 vines representing five different autochthonous cultivars were found to be infected (4.8%), giving the overall infection rate in the collection plantations of 3.6% (35/983).Screening performed in 93 commercial vineyards, 16 located in the continental region (441 vines/samples) and 77 in the coastal region (2903 vines/samples), revealed no presence of GVG in the continental area, while in the coastal region, virus was confirmed in 421 (~14.5%) grapevines from 22 locations (28.6%). Considering the individual locations, the highest incidence of GVG was found in the locations Furnaže and Marceline  and Pag 4 (Zadar County), where a total of 50, 54, and 30 grapevines of cvs. Mladenka, Vlaška and Paška maraština were GVG-positive, respectively. Besides the mentioned autochthonous cultivars, infection incidence above 50% was also found in cultivars Cipar, Ljutun and Muškatel. The lowest infection rate was found in the location Zemunik Donji (Zadar County), with 2% infections in the autochthonous cv. Plavina, although the general infection rate of cv. Plavina was 32.5%. Of the 506 analyzed vines of the cv. Plavac mali, the most popular autochthonous grape variety in Croatia, only one (0.2%) was positive for GVG. Concerning the overall number of 4327 grapevines tested in this survey, GVG infection was detected in 456 samples, representing an overall infection rate of 10.5%. Interestingly, infection was not confirmed in imported cultivars and rootstocks included in the study. A detailed overview of the sampling vineyards together with GVG-positive vines and corresponding locations can be found in Considering the regional distribution, GVG presence was confirmed in 6 of the 12 counties included in the survey. The highest infection rate was found in Zadar County—210/727 (28.9%), followed by Šibenik-Knin County (15%), Split-Dalmatia County (13%), Dubrovnik-Neretva County (4%), the City of Zagreb (3.3%) and Primorje-Gorski Kotar County (2.4%). The presence of the virus was not confirmed in other counties surveyed , as well as four newly discovered isolates originating from the Kaštela-Marceline site. Groups 1 and 5 represent a previously unknown spectrum of GVG genetic diversity, which was revealed in this study . NucleotHTS has made great progress in the discovery of viruses in many plant species, including grapevine ,23, wherAs demonstrated on five grapevine accessions, GVG can be detected by real-time RT-PCR throughout the growing season and the dormancy period using available plant tissue . In contrast, false-negative results for grapevine leafroll-associated virus 3 (GLRaV-3) ,25 and gA field survey done in commercial vineyards confirmed the presence of GVG in ~14.5% vines. Compared to similar studies done for other vitiviruses in Croatia, the occurrence of GVG is less than the determined infection with GVA (61.4%), but significantly higher compared to GVB (3.1%) . InfectiHemiptera: Pseudococcidae) and soft-scale insects (Hemiptera: Coccidae) [Phylogenetic analyses performed on the 37 newly discovered and characterized Croatian GVG isolates, and 11 previously known isolates from the GenBank, separate the isolates into five groups. While GenBank isolates from the USA and New Zealand were assigned each into distinct groups (3 and 4), isolates from Croatia were dispersed into three groups . Interestingly, Group 2 comprised previously known isolates from Croatia, together with five newly discovered isolates , all isolates in the mentioned group originating from the Kaštela region. Other newly characterized GVG isolates were classified into two different groups (1 and 5), representing novel, not known, genetic diversity of GVG. Thus, Group 5 showed to be the most abundant in the number of isolates, while Group 1 includes the largest number of isolates originating from different sites/vineyards. Another particularity noticeable from the phylogenetic tree was the grouping of isolates collected from the same site/vineyard close to each other. Such grouping suggests possible common ancestry and/or on-site transmission by insect vectors. The possibility of insect transmission could be assumed, especially since some other grapevine-infecting vitiviruses  can be successfully transmitted by various mealybugs (occidae) ,32. Addioccidae) . The Fstoccidae) . The Fstoccidae) . In contUltimately, this study is evidence of the wide distribution of GVG in Croatian autochthonous grapevine cultivars originating and/or grown along the Croatian coastal grapevine growing region. This may be important since some vitiviruses associated with RW disease can cause significant losses in grapevine production due to their effects on grapevine physiology, growth, yield, propagation, and wine quality. In previous studies, vitiviruses were often found together with economically important viruses from the leafroll complex. Viruses from these two complexes are thought to have specific interactions with each other and with the same vectors, allowing better transmission by coinfection . AccordiWith this study, we expanded the knowledge of GVG biodiversity and developed a new diagnostic tool based on the RT-PCR method that can be used in monitoring programs. Considering the confirmed GVG infection rate of 10.5% in Croatia and the proven negative impact of some other vitiviruses  on grapevine, we have created the necessary prerequisites for future research aimed at a better understanding of GVG ecology, the impact on grapevine production and, consequently, possible inclusion in certification schemes.https://primer3.org/webinterface.html, accessed on 5 August 2019) and Geneious 10.2.6  programs.For the construction of primers and probes, 11 sequences available from the GenBank , along with two GVG-positive vines from the Grapevine Virus Collection in Zagreb determined during this study, were used. To obtain insight into full-genome sequence, those two GVG-infected grapevine accessions were subjected to total nucleic acids (TNA) extraction and HTS, according to the previously described protocol [The real-time RT-PCR assay, which included 18S rRNA oligos as an internal control , was pre2CO3, 0.035 M NaHCO3, 0.0005 M PVP, 0.2% bovine serum albumin, 0.05% Tween 20, pH 9.6 with acetic acid). After centrifugation at 13,200× g for 10 min, the supernatant was transferred to a new 2 mL collection tube. In a 100 µL of GES buffer  prepared, 8 µL of extract were added and denaturized in Mastercycler  at 95 °C for 10 min. Subsequently, the availability of nucleic acids was checked spectrophotometrically on NanoPhotometer P330 .Isolation of RNA used in the above-mentioned assay was done by the previously described glycine-EDTA-sodium (GES) method  using 0.To validate the new real-time RT-PCR, a GVG-positive grapevine from the Grapevine Virus Collection was selected and subjected to RNA extraction. Sensitivity and validation comparison was performed with 10-fold serial dilutions, ranging from 1 to 100,000, in water and prepared in three replicates. The reactions consisted of 2 µL of each dilution in 20 µL of the final volume. Finally, the efficiency of the assay was evaluated using the results of a standard curve .To obtain real-time RT-PCR detection capability during active growth and dormancy, five GVG-infected grapevine accessions of the cultivar Vlaška  were selected from the Grapevine Virus Collection. Sampling and RNA isolation were performed at 16 different time points: at the beginning of vegetation from shoots (April), during vegetation from leaf petioles (mid-May to mid-November) and from cortical scrapings during dormancy (mid-November to March). Quantification cycle (Cq) values for GVG and 18S rRNA were measured and processed with the software IBM SPSS Statistics, ver. 25 . In addiThe Kolmogorov–Smirnov (KS) and Shapiro–Wilk tests were used to evaluate normality ,41. One-As part of the study on the incidence and distribution of GVG in Croatia, samples of autochthonous and introduced grapevine cultivars were collected in June/July 2020 and 2021, both from collection plantations and commercial vineyards along the Croatian continental and coastal grapevine growing regions . In the To get insight into sequence homology of different GVG isolates, conventional one-step RT-PCR assay was used; thus, primers covering almost all the coat protein (CP) region  were constructed using the data from the same sequences as for the design of primers and the probe for real-time RT-PCR. The reaction was prepared in a 25 µL mixture using a One-step RT-PCR kit , according to manufacturer’s recommendation, with the addition of 0.2 µL of isolated RNA as a template and 0.4 µM of each primer. Reactions were performed in a Mastercycler  under the following conditions: reverse transcription at 50 °C for 30 min, initial activation step at 95 °C for 15 min, 35 cycles at 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a final elongation step at 72 °C for 10 min. 1X TBE buffer was used to prepare a 1.5% agarose gel with one drop of GelRed . Lastly, amplicons were verified by horizontal gel electrophoresis and visualized on the UV Transilluminator 2000 . Thirty-five GVG-positive plants identified during the field survey and originating from 20 different vineyards/sites were selected for conventional one-step RT-PCR, followed by direct sequencing. Multiple grapevines (two or three) were sampled from nine sites  to obtain information on GVG genetic diversity within the same sites, while one grapevine was sampled from 11 sites  to obtain information on variance among sites. Sanger sequencing was performed in both directions at Macrogen Europe . The sequences were processed in Bioedit 7.2. . The 35


Colletotrichum spp. To obtain potential biological agents, we identified four novel RNA viruses and obtained full-length viral genomes from turfgrass pathogenic Colletotrichum spp. in Japan. We characterized two novel dsRNA partitiviruses: Colletotrichum associated partitivirus 1 (CaPV1) and Colletotrichum associated partitivirus 2 (CaPV2), as well as two negative single-stranded (ss) RNA viruses: Colletotrichum associated negative-stranded RNA virus 1 (CaNSRV1) and Colletotrichum associated negative-stranded RNA virus 2 (CaNSRV2). Using specific RT-PCR assays, we confirmed the presence of CaPV1, CaPV2 and CaNSRV1 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-264, as well as CaNSRV2 in dsRNAs from original and sub-isolates of Colletotrichum sp. MBCT-288. This is the first time mycoviruses have been discovered in turfgrass pathogenic Colletotrichum spp. in Japan. CaPV1 and CaPV2 are new members of the newly proposed genus “Zetapartitivirus” and genus Alphapartitivirus, respectively, in the family Partitiviridae, according to genomic characterization and phylogenetic analysis. Negative sense ssRNA viruses CaNSRV1 and CaNSRV2, on the other hand, are new members of the family Phenuiviridae and the proposed family “Mycoaspirividae”, respectively. These findings reveal previously unknown RNA virus diversity and evolution in turfgrass pathogenic Colletotrichum spp.Turfgrass used in various areas of the golf course has been found to present anthracnose disease, which is caused by  Colletotrichum spp., which induces foliar blight, basal stem rot and eventually host mortality in turfgrass species. These symptoms progress to irregular patches. Because of the importance of anthracnose disease, researchers have been motivated to conduct research on the detection, characterization and management of Colletotrichum spp. in turfgrass hosts . C. C42]. CAlphapartiti-virus with strong bootstrap support  tail region and had an ORF (ORF1) in its complementary (vc) strand. 5′- and 3′-ends of the CaNSRV1 genome were significantly similar to their respective ends in Grapevine associated cogu-like virus 3 (GaCLV3) or Grapevine laula-virus 3 (GLV3), Grapevine associated cogu-like virus 2 (GaCLV2) or Grapevine laula-virus 2 (GLV2) and Laurel Lake virus (LLV) of the Phenuiviridae family. ORF1 product consisted of 2382 aa with a predicted molecular weight of 274.0 kDa  , a strategy used by many negative-stranded viruses to translate viral proteins by using capped terminal ends from host mRNAs  was found from the 74.0 kDa A. The NC1387 aa) A. BLASTpavirales . Furtheravirales B. The most mRNAs C 50]. T. TColletus clade D, which −16) (575EREQKYEARLF586) of CaNSRV2 was 100% identical to that of FpNSRV1 and 91% identical to Cladosporium cladosporioides negative-stranded RNA virus 1 (CcNSRV1), with perfect conservation of the lysine (K), arginine (R) and glutamic acid (E) residues. The motif-A (S643LLLDIEGHNQSMQ656) of CaNSRV2 exhibited similarities with the motif A of ophiviruses, which included conserved leucine (L), aspartic acid (D), glycine (G), histidine (H), asparagine (N) and serine (S) residues and was 100% identical to FpNSRV1 and 92.8% identical to CaNSRV2. The glycine residues were conserved in motif B of CaNSRV2 and other related viruses. Motif C, found in the RdRp of CaNSRV2 and comparable viruses, contained the YSDD signature, which was also found in Orthomyxoviridae members and corresponded to the GDNQ signature found in the majority of negative-stranded RNA viruses, which acts as an RdRp active site  and RNA-2  and no poly (A) tail regions were detected in both segments A. We fou−16) A. The pu−16)  51]. Mu. Mu−16) ive site C. In motive site D. NLSs wuvirales  has reveuvirales .Another segment of the identified genome of CaNSRV2 was predicted to possess four ORFs  A. Among Colletotrichum spp. in Japan in order to develop bio-control agents in the future. Although several viruses have been identified in Colletotrichum spp. isolated from other host plants, no mycoviruses have been reported to date in Colletotrichum spp. pathogenic to turfgrass. Using screening employing dsRNA electrophoresis combined with FLDS, we successfully identified four new RNA viruses from Colletotrichum spp. infecting turfgrass in Japan  of the genus Alphapartitivirus has been linked to the color of fruiting bodies of the host fungus [Although members of the ir hosts ,42,53, at fungus ,55,56. Nt fungus . It remaColletotrichum sp. MBCT-264. We proposed that CaPV2 is a new species of the genus Alphapartitivirus based on the sequence identities of RdRp and CP genes with other members of the genus. Alphapartitiviruses typically have two dsRNA segments of similar size, but CaPV2 was discovered to have four genome segments, with only the RdRp- and CP-encoding segments showing considerable sequence similarity to other partiti-viruses  that mycophio-viruses might have extra genomic segments associated with them, which has yet to be confirmed [We also discovered CaNSRV2 from d others ,51,62. Tonfirmed . CaNSRV2As mentioned above, neither CaNSRV1 nor CaNSRV2 have MP or Nc, although MP may not be required for their replication in their fungal host. Given that nucleocapsid formation is a requirement for (−) ssRNA virus replication, it will be interesting to investigate how viruses such as CaNSRV1 or CaNSRV2 replicate in their fungal hosts.Colletotrichum sp. MBCT-264. Multiple viral infections are common in fungi [R. necatrix isolate, Chun and colleagues reported the presence of a fusagravirus, Rosellinia necatrix fusagra-virus 4 and its co-infection with a partiti-virus, Rosellinia necatrix partiti-virus 26 (RnPV26) [R. necatrix strain W442 was reported to co-infect with the partitiviruses RnPV18 (of the genus Betapartiti-virus) and RnPV19 (of the genus Alphapartiti-virus) [Colletotrichum sp. MBCT-264 interact with one another and have any effect on the host is unknown. Our findings showed that a single Colletotrichum strain could be infected with multiple mycoviruses. We need to observe mycovirus diversity in other Colletotrichum spp. that infect grasses and other gramineous plants since it is interesting to see distinct patterns of virus infection in Colletotrichum spp. MBCT-264 and Collectotrichum sp. MBCT-288. Further research into the biological effects of the mycoviruses discovered in this study is required, because we did not perform horizontal transmission experiments in this study. Identifying hypovirulent mycoviruses and determining their potential use in biocontrol programs is appealing because reducing the impact of Colletotrichum species in turfgrasses is important for maintaining the green beauty of golf fields. It is also worth looking into the role of mycoviruses on turfgrasses–Colletotrichum interactions, as this could reveal new information about the pathogenicity mechanism of Colletotrichum spp.Furthermore, we report the coexistence of two partitiviruses and a negative-stranded ssRNA virus in in fungi ,69 and ain fungi ,70. In a(RnPV26) . The Japi-virus) . Virusesi-virus) ,72. Whet


Little information is available on the costs of respiratory syncytial virus (RSV) in Vietnam or other low- and middle-income countries. Our study estimated the costs of LRTIs associated with RSV infection among children in southern Vietnam.We conducted a prospective cohort study evaluating household and societal costs associated with LRTIs stratified by RSV status and severity among children under 2 years old who sought care at a major pediatric referral hospital in southern Vietnam. Enrollment periods were September 2019–December 2019, October 2020–June 2021 and October 2021–December 2021. RSV status was confirmed by a validated RT-PCR assay. RSV rapid detection antigen (RDA) test performance was also evaluated. Data on resource utilization, direct medical and non-medical costs, and indirect costs were collected from billing records and supplemented by patient-level questionnaires. All costs are reported in 2022 US dollars.536 children were enrolled in the study, with a median age of 7 months (interquartile range [IQR] 3–12). This included 210 (39.2%) children from the outpatient department, 318 children (59.3%) from the inpatient respiratory department (RD), and 8 children (1.5%) from the intensive care unit (ICU). Nearly 20% (105/536) were RSV positive: 3.9 percent (21/536) from the outpatient department, 15.7% (84/536) from the RD, and none from the ICU. The median total cost associated with LRTI per patient was US$52 (IQR 32–86) for outpatients and US$184 (IQR 109–287) for RD inpatients. For RSV-associated LRTIs, the median total cost per infection episode per patient was US$52 (IQR 32–85) for outpatients and US$165 (IQR 95–249) for RD inpatients. Total out-of-pocket costs of one non-ICU admission of RSV-associated LRTI ranged from 32%-70% of the monthly minimum wage per person (US$160) in Ho Chi Minh City. The sensitivity and the specificity of RSV RDA test were 88.2% (95% CI 63.6–98.5%) and 100% (95% CI 93.3–100%), respectively.These are the first data reporting the substantial economic burden of RSV-associated illness in young children in Vietnam. This study informs policymakers in planning health care resources and highlights the urgency of RSV disease prevention.The online version contains supplementary material available at 10.1186/s12879-023-08024-2. Acute lower respiratory tract infections (LRTIs) are a major public health problem for children in Vietnam and in all low- and middle-income countries (LMICs). Respiratory syncytial virus (RSV) is the leading cause of LRTIs in pediatric populations. Epidemiological studies have consistently shown high rates of severe RSV infection in children between 1 and 5 months of age, with 99 percent of deaths occurring in LMICs where 90% of children under 5 years old live , 3. A poThe RSV season in South Vietnam typically occurs between May and November, during the rainy season –4. ChildThere is currently no licensed RSV vaccine. Passive prophylaxis for RSV is available through palivizumab , a neutralizing anti-RSV fusion protein monoclonal antibody (mAb), but due to its high cost, palivizumab is not available or has minimal access in Vietnam and other LMICs .Among the 40 recent RSV vaccine and mAb candidates, the long-acting mAb Nirsevimab™  is the most advanced and has shown encouraging results in Phase 2b and 3 trials . NirseviA robust estimate of RSV-related health burden and cost will help evaluate the potential impact and cost-effectiveness of future RSV prevention and inform national procurement and prioritization decisions. To our knowledge, no information is available on the costs of RSV in Vietnam and very little is available for other LMICs in Southeast Asia . AlthougWe present here estimates of direct medical and non-medical costs and indirect costs of LRTIs associated with RSV infections among children visiting for consultations at outpatient departments or hospitalized at CH1. In this study, we also evaluated the performance of the RSV RDA test Immuno AG1 in a subset of patients.A prospective cohort study was conducted at the outpatient and inpatient departments of CH1, a tertiary pediatric care facility previously involved in studies assessing LRTIs and RSV in HCMC, Vietnam .In general, patients with LRTIs, defined as cough or difficulty breathing , initialWe evaluated the costs associated with LRTIs, stratified by RSV status and severity, among children under 2 years of age seeking care at CH1.Mild: patients admitted only to the outpatient department2) at admission ≥ 93 percentModerate: patients admitted to the respiratory department and who had saturation oxygen (SaO2) < 93 percentSevere: patients admitted to the respiratory department and who had saturation oxygen at admission  recommendations  as belowChildren were recruited to our study prospectively. Only patients who met all inclusion criteria and did not have any exclusion criteria were enrolled. Inclusion criteria were having cough and/or difficulty breathing, a clinical diagnosis of LRTI based on WHO criteria , and onsWe used the formula below, based on WHO guidelines for estimating treatment costs for diarrheal disease , to calcIn the formula, CV is the coefficient of variation and N is the annual case load. Annual case loads for both outpatient and inpatient calculations were based on historical data from CH1 regarding annual LRTI admissions  swab was collected from each study participant, within 24 h of study enrollment, to determine RSV infection status using a validated real-time RT-PCR (reverse transcription polymerase chain reaction) test . NPs forA random sample of 30 percent of participants admitted to the respiratory ward department also had an additional NP swab . This additional NP swab was tested immediately on-site via a commercial RSV RDA test .Although the physicians who treated patients were not aware of RSV RT-PCR results, they received results from the RDA test in real-time. All laboratory technicians who conducted the RT-PCR testing were blinded about the RSV RDA results.Costs were estimated from the societal and household perspectives. Three components of cost and resource utilization data were collected for the study:(1) Health care utilization and cost data from facility-level billing data.(2) A day-of-visit parent questionnaire to assess household-level costs incurred prior to arriving at the facility. For outpatient participants, any costs due to seeking care incurred on the day of the visit were also collected.(3) A post-discharge parent questionnaire to identify additional costs incurred by the household for the same illness 1 week after discharge from the facility.The billing data were used to identify resource utilization as well as direct medical and overhead costs associated with care received in the facility. Data parameters collected include types of services received during the visit , consultation fees, medications, laboratory tests, imaging , and other procedures . The proportion of costs covered by the household and national health insurance system (NHIS) was also reflected on facility-level billing data. Consumption of health care was the direct medical cost that covered the total costs of disease episode.The total costs per case from the societal perspective including direct medical costs (from both billing and questionnaire data), non-medical costs, and indirect costs incurred prior to admission, during the visit and after discharge.The household-level costs included patient-level out-of-pocket, non-medical costs , and indirect costs  during the disease course. These costs were obtained through standardized questionnaires conducted with parents (pre- and post-discharge), as described above.Hence, we reported:We also collected data on total costs, stratified by time of spending, using a specific questionnaire for each spending period. Pre-hospital costs include direct medical costs, non-medical costs, and indirect costs estimated from household surveys for any health care services sought before the day of outpatient visit or inpatient/ICU admission. Costs incurred during the hospital stay include direct medical costs from billing data plus non-medical and medical out-of-pocket spending  for the day of outpatient visit or the episode of inpatient stay, as well as indirect costs incurred during the disease episode. Similarly, follow-up visit costs include direct medical costs from billing data for follow-up visits at the hospital, as well as out-of-pocket spending and indirect costs for any care sought after the day of outpatient visit or discharge until completing a follow-up visit.The NHIS covers a certain proportion of the direct medical cost, depending on the type of medicines and procedures received during the hospitalization or the visit. The NHIS coverage amount was reflected on the billing data collected for the study. The remaining cost was from the out-of-pocket expenditures by the patient’s family .Demographic characteristics of children and their families were also collected through questionnaires for both outpatients and inpatients.Figure Costs were estimated by the following categories: cost per episode, cost by component , and cost by RSV status and severity .In calculating indirect costs, opportunity costs of lost income due to missed work were based on the average salary reported by participants in the study, and opportunity costs of lost leisure time were based on the minimum Vietnamese income based on government data, for the study period . ContinuWe also evaluated sensitivity, specificity, and positive and negative predictive value of the RSV antigen rapid test  and calculated 95 percent CIs. Statistical comparisons between the RSV antigen rapid test and the validated real-time RT-PCR were performed using McNemar’s test .All statistical analysis was implemented using Stata version 16.0 (StataCorp LP). All statistical tests were conducted at the two-tailed 5 percent significance level.We enrolled 537 children in our study. One case was discarded from the final analysis as more than 50 percent of the relevant data were missing, including the billing cost and the questionnaire data; thus, the full analysis was conducted on 536 participants. This included 210 children (39.2 percent) from the outpatient department; 318 children (59.3 percent) from the respiratory department; and 8 children (1.5 percent) from the ICU. Demographic and clinical characteristics of patients are shown in Table The median total cost from the societal perspective per LRTI episode (regardless of RSV status) per patient was US$52 (IQR 32–86) for outpatients and US$184 (IQR 110–287) for inpatients (not including ICU patients). As the costs of ICU cases were substantial compared to the costs of other inpatients, we estimated the total costs of ICU cases separately. The total cost per ICU admission was an 11-times the total cost per non-ICU admission  for outpatients and US$165 (IQR 95–249) for inpatients  for outpatients and US$190 (IQR 123–292) for inpatients. The median total cost per disease episode for RSV-negative cases was significantly higher than the total cost of RSV-positive cases  for inpatients. Specifically, the direct medical cost, in-hospital cost, and follow-up cost for non-RSV LRTI cases were significantly higher than those for RSV-LRTI cases (Table Approximately 1 percent (3/210) and 86.8 percent (276/318) of outpatients and inpatients were covered by the NHIS, respectively.The median total household cost per LRTI period per patient was US$51 IQR 32–85) for outpatients and US$112 (IQR 58–184) for inpatients (not including ICU patients). The total household cost per ICU admission was a 5-times the total household cost per non-ICU admission (Table 2–85 for Most families of outpatients (87.1 percent), non-ICU inpatients (86.8 percent), and ICU patients (100 percent) reported having to use their savings to cover disease treatment and lost income Table .For RSV-associated LRTI cases, the median total household cost per infection per patient was US$52 IQR 32–85) for outpatients and US$117 (IQR 65–183) for inpatients . Among inpatients, significant differences in the antibiotic, laboratory cost, imaging and procedure costs were observed between the RSV and non-RSV groups  Table . Non-RSVIn total, we had 70 cases tested by both RSV RDA and RSV real-time RT-PCR. The number of RSV detections by RSV RDA test (Immuno AG1) test was not significantly lower than by real-time RT-PCR (15/70 [21.4 percent] versus 17/70 [24.3 percent), McNemar test p-value = 0.5). Table Based on the results shown in Table Our study showed the costs associated with LRTIs are substantial and somewhat higher among children with RSV-negative status and increased severity of disease. For outpatient visits, a patient’s family usually paid out-of-pocket for examination and treatment. Inpatients less than 5 years of age are covered by the NHIS at 80 percent of the costs of listed medications and interventions if they follow their catchment area for hospitalization, are admitted due to an emergency, or are formally referred to a tertiary public hospital. Despite this coverage, the total out-of-pocket cost for outpatients and non-ICU inpatients for one LRTI event were still significant, representing about 32 to 70 percent of the monthly minimum wage per person ($160)  in HCMC.During the first year of life, incidences of acute lower respiratory infections (ALRI) leading to outpatient visit and hospitalization were previously reported to be 461 and 81 per 1000 children, respectively . Among cBecause the clinicians in our study were not blinded for the RSV RDA result, this could have influenced their decisions on the treatment and management for those cases. We compared the demographics, clinical characteristics, societal costs, out-of-pocket costs and the consumption of health care system costs between patients having their sample processed with the RDA test and those who did not have the RDA test  pneumoniae [S. pneumoniae carriage has been previously reported to significantly increase the S. pneumoniae load and be associated with radiologically confirmed pneumonia [In our study, other pathogens (non-RSV status) and severity were the two factors significantly associated with the total costs Table , specifieumoniae . RSV co-neumonia , which lneumonia .Of note, it has been shown that the incidences of severe RSV cases and associated deaths have been disproportionally reported in LMICs such as Vietnam . The higThere are some limitations in our study that should be acknowledged. First, our study was conducted during the COVID-19 pandemic, limiting the patient enrollment and interrupting data collection. The usual RSV season also changed during COVID-19 and became hard to predict. It was not clear if RSV infections during COVID-19 pandemic are less severe than pre-pandemic infections. We did not see differences in severity but cannot make conclusions as our sample size was small. Although we met our lower-bound target for the number of children to be enrolled (based on an expected 30 percent RSV-positivity rate), the overall number of RSV cases in our study was lower than anticipated (19 percent) and, thus, we did not reach the estimated sample sizes for RSV-positive cases, limiting the certainty of these cost estimates. Second, we did not have RSV cases among ICU patients. We were able to characterize non-RSV ICU costs, but we cannot draw concrete costs for very severe RSV cases. Hence, this study does not allow us to describe the whole spectrum of costs associated with RSV disease. Third, we did not record information about number of eligible patients across the facility due to high hospital volumes and, therefore, are not able to characterize the difference between eligible LRTI patients and those enrolled in our study. This limits our ability to evaluate potential selection bias and generalizability of the study. Fourth, our costs relied on billing data, which often reflect charges to the patient or payer and may or may not reflect the true costs of resource utilization. Finally, we did not have the information about the gestational age of patients in our study, we cannot estimate what the impacts of the preterm births on RSV cost. Compared to term infants, preterm infants have a 2- to threefold higher hospitalisation rate , 32 and This study provides, for the first time, the complete costs (direct and indirect) associated with LRTIs in Vietnam in general and with RSV-LRTIs specifically. These costs are substantial, leading to significant economic burden to families and potentially to the health care system and country as the NHIS covers about 80 percent of the direct medical cost. These cost estimates provide valuable data for economic analysis of LRTI management and preventive strategies generally and for LRTIs associated with RSV.Additional file 1. Additional Tables.


Cucumovirus, family Bromoviridae), in a plant pathogenic basidiomycetous fungus, Rhizoctonia solani, isolated from a potato plant grown in the field. Here, we further extended the study to investigate whether similar cross-infection of plant viruses occurs widely in plant-associated fungi in natural conditions. Various vegetable plants such as spinach, leaf mustard, radish, celery, and other vegetables that showed typical virus-like diseases were collected from the fields in Shandong Province, China. High-throughput sequencing revealed that at least 11 known RNA viruses belonging to different genera, including Potyvirus, Fabavirus, Polerovirus, Waikavirus, and Cucumovirus, along with novel virus candidates belonging to other virus genera, infected or associated with the collected vegetable plants, and most of the leaf samples contained multiple plant viruses. A large number of filamentous fungal strains were isolated from the vegetable leaf samples and subjected to screening for the presence of plant viruses. RT-PCR and Sanger sequencing of the PCR products revealed that among the 169 fungal strains tested, around 50% were carrying plant viruses, and many of the strains harbored multiple plant viruses. The plant viruses detected in the fungal isolates were diverse (10 virus species) and not limited to particular virus genera. However, after prolonged maintenance of the fungal culture in the laboratory, many of the fungal strains have lost the virus. Sequencing of the fungal DNA indicated that most of the fungal strains harboring plant viruses were related to plant pathogenic and/or endophytic fungi belonging to the genera Alternaria, Lecanicillium, and Sarocladium. These observations suggest that the nonpersistent acquisition of plant viruses by fungi may commonly occur in nature. Our findings highlight a possible role for fungi in the life cycle, spread, and evolution of plant viruses.Investigating a virus’s host range and cross-infection is important for better understanding the epidemiology and emergence of viruses. Previously, our research group discovered a natural infection of a plant RNA virus, cumber mosaic virus (genus  Plant viruses cause major losses in agricultural production worldwide by reducing crop yields and/or the quality of agricultural products . NeverthPotexvirus, family Alphaflexiviridae) have been isolated from ascomycete fungi, Botrytis cinerea, and Sclerotinia sclerotiorum ) belonging to the family Solemoviridae and a polerovirus-associated subviral RNA (turnip yellows virus-associated RNA [TuYV-aRNA]), one fabavirus , one waikavirus , one umbravirus , and CMV. Selected virus-related sequence contigs have been deposited to the GenBank/EMBL/DDBJ databases  derived from the RNA-seq data set  revealed numerous virus-related sequences . As listatabases . These patabases . BesidesLuteoviridae) [Enamovirus. QRV2-encoded putative RdRP had a 48.3% identity with that of beihai narna-like virus 23, a monopartite narna-like virus [Phenuiviridae [Rubodvirus. QRV4 had a negative-strand genome  to known viruses and, therefore, were provisionally identified as Qingdao RNA virus 1–4  ,40. QRV1ke virus . The preke virus . QRV3 haiviridae ,44. A sed genome , and its insects . Moreovesequence  suggest Fifteen virus-related contigs representative of each viral genome or segment were selected for further analyses, considering their length (>2000 nt) and composing read number (>1000 reads) as well as their unrelatedness to bacterial viruses . RT-PCR Representative leaf tissue of each sample was used as the source material for the isolation of fungal strains. A total of 241 fungal strains were isolated, with the number of fungal strains obtained largely differing among the leaf samples . UnfortuCucumovirus, Potyvirus, Polerovirus, Waikavirus, Rubodvirus, Qinvirus, Fabavirus, and Enamovirus) were detected in the fungal strains, whereas KoMV, QRV2, and WMV were not detected in any of the fungal strains. CMV was most frequently detected in the fungal strains, with 38.4% prevalence (43 out of 112 strains tested), which is in accordance with the observation that CMV was present in the majority of the leaf samples  , but their species classification was not clear  , while tot clear . S. kiliored CMV . Thus, iPartitiviridae), alphaflexiviruses (non-segmented (+)ssRNA viruses, family Alpha-flexiviridae), and endornaviruses (capsid-less (+)ssRNA viruses, family Endornaviridae) [Diverse viruses that infect fungi  have been identified in various groups of fungi ,45,46. Aviridae) ,47,48. TBromoviridae), was the most frequently detected in the fungal strains. This result seems plausible as the majority of the leaf samples were infected with CMV. CMV has the largest host range among plant viruses and is one of the most widespread plant viruses in agricultural fields [R. solani was previously discovered [Potyviridae), and BBWV2, a fabavirus (family Secoviridae), were reported to be the predominant viruses infecting vegetable crops in China [Polerovirus (family Solemoviridae), has been reported to infect crucifer crops in China and Japan [Secoviridae), was first identified in a transcriptome dataset of rapeseed [Enamovirus and Rubodvirus, respectively, virus genera that consist of plant-infecting members [Lentinula edodes) [In this study, we identified ten genetically diverse viruses representing eight virus genera and six virus families carried by fungal strains isolated from the leaves of diseased plants. Notably, most of these viruses  are known to be vectored by insects, such as aphids and leafhoppers . Thus, ol fields . As natuscovered , this stin China . ZYMV, ain China ,52,53,54nd Japan ,56. TuYVnd Japan ,58. Althrapeseed . QRV1 an members ,61, sugg members ,62,63. T edodes) , suggestS. kiliense, L. coprophilum, and Alternaria sp., the majority being S. kiliense. Genus Sarocladium is comprised of a highly diverse group of fungi including plant pathogens, endophytes, saprobes, mycoparasites, and human opportunistic pathogens [S. kiliense is mainly known as a human opportunistic pathogen [S. kiliense strains isolated in this study against the nucleotide collection in GenBank yielded the majority of hits representing endophytes isolated from plant samples [L. coprophilum was first identified from the fresh fecal matter of Marmota monax (groundhog) [Lecanicillium are known to be entomopathogens, mycoparasites, or endophytes [Alternaria species are ubiquitous in the environment, with a number of species being major plant pathogens, saprophytes, endophytes, and opportunistic pathogens or allergens of humans [The fungal strains in this study that were infected with plant viruses belonged to three fungal species, athogens ,66. S. kpathogen ,68,69,70pathogen ,73,74,75 samples  (see Supoundhog) . The spedophytes ,79. Altef humans ,81,82. AOphiovirus (family Ophioviridae), Varicosavirus (family Rhabdoviridae), Potexvirus (family Alphaflexiviridae), and several genera in the family Tombusviridae are transmitted by the zoosporic soil-inhabiting fungi Olpidium virulentus, O. brassicae, and O. brassicae (family Olpidiaceae), although viruses do not multiply in these fungal vectors [R. solani, CMV is horizontally transmitted by hyphal anastomosis but not vertically transmitted through basidiospores , while in F. graminearum, TMV is vertically transmitted through conidia  [The finding of the common acquisition of plant viruses by plant-associated fungi highlights the possibility that fungi are involved in the epidemiology, emergence, and evolution of plant viruses. In an attempt to understand the epidemiology and emergence of plant viruses, intensive investigations have been conducted on the vector transmission and host range or reservoir of plant viruses in nature ,83,84; h vectors . The bio spores) ,22. Thes


Over the past seven years, the global community of clinicians, researchers, and public health professionals have collaboratively discovered and disseminated knowledge about the congenital Zika virus infection, which was virtually unknown prior to 2015. It is remarkable to reflect upon the progress made in this relatively short time, from the initial emergence of a “new” congenital infection, which was potentially devastating to unborn babies, to our current much deeper understanding of this disease. It is a testament to the many people that have engaged in focused research, clinical care, and advocacy regarding congenital Zika virus infection. This research is being conducted all over the world as we now seek to understand long-term childhood outcomes of Zika virus. From the Zika virus epidemic, many important lessons were learned about fetal infection, the risk to offspring neurodevelopment and importance of health surveillance and global partnerships, which are applicable to many other diseases affecting fetuses and newborns. Due to a heightened awareness of the impact infections could have on the pregnant mother–fetal dyad because of the recent Zika virus epidemic, there was an early recognition of the potential threat to an unborn child of a mother with SARS-CoV-2 infection in pregnancy during the current COVID-19 pandemic. During the past seven years, our understanding of the diagnosis and limitations of Zika virus diagnostic testing has improved. We have learned how to evaluate pregnant women, infants, and children for neurologic and other organ system abnormalities associated with Zika virus exposure, and we are now just fully beginning to appreciate the range of long-term neurodevelopmental outcomes associated with prenatal Zika virus infection ,2,3,4. ITropical Medicine and Infectious Disease, entitled ‘Zika in Infants and Children’, we present an outstanding collection of ten manuscripts that contribute to our enhanced knowledge of Zika virus in infects and children. The manuscripts describe patients from Brazil, Colombia, Honduras, Thailand, the United States (U.S.) Virgin Islands, and from the World Health Organization’s (WHO) Zika Individual Participant Data Meta-Analysis (IPD-MA), which includes data from 28 countries and territories [In this Special Issue of ritories . The theritories , infant ritories ,7,8, indritories , postnatritories , ocular ritories , importaritories , surveilritories ,6,10,12,ritories ,13.In a cohort of pregnant women with symptomatic Zika virus infection during pregnancy in Colombia, 4.2% of fetuses/infants were found to have Zika-associated brain or eye defects, including microcephaly at birth, which is comparable to other published cohorts . In the Much research has focused on congenital infection for Zika virus infections in infants and children. However, the infant brain experiences important phases of development postnatally and throughout early childhood. In the review article by Drs. Jessica Raper and Ann Chahroudi, emerging clinical evidence supports the hypothesis that Zika virus infection during early infancy can result in negative neurologic consequences . Animal The effective study of an emerging global infectious disease requires robust international research partnerships. The Zika virus IPD-MA is currently harmonizing data from over 52 cohort studies from 28 countries and territories to determine the risk of adverse pregnancy and infant outcomes, risk on childhood outcomes, and to enable a plan to address a future Zika virus epidemic . While tSome areas with consistent knowledge gaps are related to an understanding of differences in pregnancy and infant outcomes. Prenatal viral infection is complex, with many factors likely impacting outcomes, including viral virulence, the timing of infection, as well as host genetic responses and environmental factors. In a review article, Drs. Kousa and Hossain describe how prenatal viral infection can lead to a spectrum of adverse neurodevelopmental conditions or fetal demise and how phenotypic variability between cases may arise following prenatal viral infections . Their mWhile the epidemic circulation of Zika virus has ended for now, we must continue to expand our knowledge about the women whose pregnancies were impacted by Zika virus infection and their infants and children who are now growing up, so that we can best support their current and future needs. We must also remain vigilant and maintain our preparedness to care for individuals impacted by future epidemics. Travelers to geographic locales with Zika virus transmission, such as Thailand, should remain aware of the potential for Zika virus infection and use precautions to avoid mosquito bites . Zika vi


Innate immunity, especially mediated by interferons (IFNs), is regarded as the first line of host immune protection against viral infection. As a strict intracellular parasite, the virus can enter the host cell through its receptor, thus surviving and proliferating in the cell Figure 1in vitro or in vivo to provide insights into viral pathogenesis, the scientific basis for novel vaccine design, and new targets for antiviral drug development.The goals of this collection are focused on studies of the complex interactions between ISGs and viruses. It is of great significance to elucidate the function and molecular mechanisms of action of known or new ISGs Song et al. performed differential transcriptomics. As a result, 90 shared upregulated ISGs were identified in porcine IPEC-J2 cells single or coinfected with porcine epidemic diarrhea virus (PEDV) and/or transmissible gastroenteritis virus (TGEV), among which 27 ISGs have antiviral activities. Furthermore, by analyzing many references and databases, they depicted the activity of ISGs in inhibiting or delaying virus proliferation at different stages of the virus cycle, indicating potential candidate targets for antiviral research. Moreover, swine IFITM3, one of the ISGs, can significantly inhibit the infection of PEDV, TGEV, and vesicular stomatitis virus (VSV) in different cells, indicating that it has broad-spectrum antiviral activity .To explore the expression of ISGs against viral infection, In addition, Ding et al. conducted a transcriptome analysis of primary macrophages induced by IFN-γ, a type II interferon that exhibits not only antibacterial but also antiviral activity in the Chinese sturgeon (Acipenser sinensis). A total of 4004 differential expression genes (DEGs) were enriched in the GO annotation, many of which were involved in immune-related processes. KEGG enrichment analysis showed that six signaling pathways are related to interferon regulation, emphasizing the importance of IFN-γ. Wang et al. evaluated the proteome in lung tissues of a cynomolgus monkey model in the early stage of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. They identified 44 proteins from all 194 significantly altered proteins, most encoded by ISGs. These results strongly indicate that IFN-ISG-mediated signaling pathways are essential in host resistance to viral infection. Besides, Dang et al. also established a high-through screening type I interferon-inducible antiviral effectors platform based on CRISPR/Cas9 knockout library with 1908 sgRNAs targeting 359 ISGs. Using VSV-eGFP as a model virus, a subset of the highest-ranking candidates, including known or novel host factors, were identified, demonstrating useful and promising methods to screen novel ISGs against a panel of porcine viruses.Lang et al. provided a macroscopic overview of the characteristics, expressions, and mechanisms of ISGs in the central nervous system (CNS) and neurological diseases, including neurological inflammation, neuropsychiatric disorders, and neurodegenerative diseases, during viral infection. The authors also summarized up-to-date progress on viral infection in neurological disorders, exhibiting the broad promise application vision and high commercial value of ISGs as potential clinical biomarkers and therapeutic targets. Furthermore, IFITM3, a well-studied ISG, can inhibit a diverse range of pathogenic virus infections in vivo or in vitro. But the precise antiviral mechanisms remain unclear. Wen et al. reviewed the latest progress on the antiviral mechanism of IFITM3, the regulation mechanism by four post-translational modifications (PTMs), especially S-palmitoylation modification, providing helpful information for understanding IFITM3 antiviral mechanism and aiding in the design of therapeutics. An et al. reported that sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1), another ISG, inhibited lipid bio-metabolic pathway to suppress flavivirus infection. Further studies showed that SAMHD1 could negatively regulate the expression of sterol regulatory element binding protein (SREBP1) and the formation of histidine aspartic acid-containing domain (HD), two critical factors associated with the lipid bio-metabolic pathway. These results revealed a novel mechanism of SAMHD1 and laid a new target for targeting lipid bio-metabolic pathway against the Flaviviridae family and other lipid-dependent viruses.As mentioned above, research on the functions and mechanisms of ISGs is crucial. in vivo models or human studies is highly summoned beyond mechanism. It may take more effort and time to get there, but it is hoped that many scientists will pay more attention to this field, solving and unveiling the most sophisticated antiviral defense systems to apply these ISGs in transgenic animal breeding or gene therapy as soon as possible.In summary, more and more antiviral ISGs have been discovered and studied with new technologies, such as unbiased screening approaches, differential transcriptome and proteome, affinity chromatography-mass spectrometry, proximity interactome, etc. The articles on this Research Topic provided new insights into the recent advances in ISGs. However, for most ISGs, the exact mechanisms of virus inhibition have not been clarified. Additionally, the vital importance of evaluating the effectiveness and safety of these ISGs LR, JB, and CL wrote the draft and revised it critically for important intellectual content. All authors contributed to the article and approved the submitted version.


What is the association of firearm assaults with US hospital costs and deaths compared with nonfirearm assaults?This cross-sectional study, using 3 years of US hospital admission and national US cause of death data on 2.4 million emergency department visits and 184 040 inpatient admissions for assaults, estimated that firearm assaults had the highest national death rates and hospital case-fatality rates compared with nonfirearm assaults. Initial hospital costs were also higher for firearm assault emergency department visits and inpatient admissions.The findings of this study suggest that it may be beneficial for policies aiming to reduce the costs of firearm violence to consider violence more broadly to understand the extent to which costs can be avoided. Estimates of the total economic cost of firearm violence are important in drawing attention to this public health issue; however, studies that consider violence more broadly are needed to further the understanding of the extent to which such costs can be avoided.To estimate the association of firearm assaults with US hospital costs and deaths compared with other assault types.International Statistical Classification of Diseases, Tenth Revision, Clinical Modification codes. Differences in ED and inpatient costs (2020 US dollars) across mechanisms were estimated using ordinary least-squares regression with and without adjustments for year and hospital, patient, and injury characteristics. The Centers for Disease Control and Prevention underlying cause of death data were used to estimate national death rates and hospital case-fatality rates across mechanisms. Cost analysis used a weighted sample. National death rates and hospital case-fatality rates used US resident death certificates, covering 976 million person-years. Hospital case-fatality rates also used nationally weighted ED records covering 2.7 million admissions. Data analysis was conducted from March 1, 2021, to March 31, 2022.The 2016-2018 US Nationwide Emergency Department Sample and National Inpatient Sample, Healthcare Cost and Utilization Project were used in this cross-sectional study of emergency department (ED) and inpatient admissions for assaults involving a firearm, sharp object, blunt object, or bodily force identified using The primary exposure was the mechanism used in the assault.Emergency department and inpatient costs per record. National death rates and hospital case-fatality rates.Overall, 2.4 million ED visits and 184 040 inpatient admissions for assault were included. Across all mechanisms, the mean age of the population was 32.7  years in the ED and 36.4  years in the inpatient setting; 41.9%  were female in the ED, and 19.1%  of inpatients were female. Most assaults recorded in the ED involved publicly insured or uninsured patients and hospitals in the Southern US. Emergency department costs were $678  for bodily force, $861  for blunt object, $996  for sharp object, and $1388  for firearm assaults. Corresponding inpatient costs were $14 702  for bodily force, $17 906  for blunt object, $19 265  for sharp object, and $34 949  for firearm assaults. National death rates per 100 000 were 0.04  for bodily force, 0.03  for blunt object, 0.54  for sharp object, and 4.40  for firearm assaults. Hospital case fatality rates were 0.01%  for bodily force, 0.05%  for blunt object, 1.05%  for sharp object, and 15.26%  for firearm assaults. In regression analysis, ED costs for firearm assaults were 59% to 99% higher than costs for nonfirearm assaults, and inpatient costs were 67% to 118% higher.The findings of this study suggest that it may be useful for policies aimed at reducing the costs of firearm violence to consider violence more broadly to understand the extent to which costs can be avoided. This cross-sectional study compares the hospital costs of various mechanisms of physical assault. Available evidence on the relative cost of firearms has shown the substantially higher health care costs compared with, for example, stab wounds.22Research into firearm violence often focuses on firearm-related deaths, in part owing to information gaps on nonfatal firearm injuries.International Statistical Classification of Diseases, Tenth Revision–Clinical Modification (ICD-10-CM) codes.23 We hypothesized that firearm assaults are associated with significantly higher costs and fatalities compared with nonfirearm assaults.We focused on a wider range of mechanisms used in assaults to examine the cost of violence in the US more broadly while further contextualizing the cost of firearm violence. We estimated emergency department (ED) and inpatient costs during initial hospital encounters, national death rates (NDRs), and hospital case-fatality rates (HCFRs) due to an assault involving a firearm compared with the other main mechanisms of violent crime  listed by the Federal Bureau of Investigation and broadly corresponding to ICD-10-CM codes; a full data set documentation has been published.25 Data analysis was conducted from March 1, 2021, to March 31, 2022. The study conforms with the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) reporting guideline for cross-sectional research studies and was deemed exempt from ethics approval by the Queen’s University Belfast Faculty Research Ethics Committee/Institutional Review Board because anonymized data were used.In this cross-sectional study, we used the US Nationwide Emergency Department Sample (NEDS) and National Inpatient Sample (NIS), Healthcare Cost and Utilization Project (HCUP), Agency for Healthcare Research and Quality discharge data from 2016 to 2018 because 2016 was the first full year to use 26 We retrospectively identified records for a firearm , sharp object (code X99), blunt object , or bodily force (code Y04) assault from the Centers for Disease Control and Prevention ICD-10-CM external cause of injury matrix.27 Suicide, unintentional, undetermined, and legal intervention injuries were excluded to provide a more consistent comparison of assailant intent and a more targeted policy discussion. Assaults are the most common intent for hospital-reported firearm injuries.28 Records involving more than 1 mechanism were few (<0.5%) and were excluded from the analysis, as were sequelae of injuries.The Federal Bureau of Investigation classifies aggravated assaults, robberies, and murders according to those in which the assailant is armed  and unarmed.29 and inflated them to 2020 US dollars using the Personal Consumption Expenditures–Hospital Care Index from the Bureau of Economic Analysis.30 Descriptive statistics of the patient and hospital characteristics related to each record for which hospital, patient, and cost data were available were calculated according to mechanism. Total cost and cost per record for ED and inpatient admissions were calculated across mechanisms. Emergency department and inpatient costs were log transformed to estimate the proportionate difference in costs across mechanism relative to firearms using ordinary least-squares regression. Models were run with and without adjustment for factors that may be related to health outcomes, the likelihood of being in an assault, or assailant intent.21 These factors were patient , injury (location of the injury or multiple injuries), hospital  characteristics, and year. Race and ethnicity  data were available as a single variable in the NIS and, as exposure to firearm violence in the US varies by race,33 we adjusted for this variable when examining inpatient costs; these data are not present in the NEDS. Location of injury was categorized using the injury mortality diagnosis matrix for ICD-10 codes  available from the Centers for Disease Control and Prevention.34 For variables with less than 5% missing data, we used a complete-case analysis,35 which was the case for inpatient costs and HCFRs. If 5% or more of the data were missing, we examined whether missingness was as good as random; if this was not the case, multiple imputation methods were used  across assault mechanisms identified using ICD-10-CM codes. To estimate HCFRs, we followed the method used by Kaufman et al17: (1) identifying the number of fatal assaults  in the WONDER database by mechanism, (2) identifying the number of nonfatal initial ED admissions using NEDS , and (3) calculating the proportion of fatal to total  cases. Because assaults involving sports equipment (code Y08.0) were not separable from assaults by other means  in the WONDER database, we excluded Y08 codes from our estimates of blunt object assault HCFRs and NDRs. In sensitivity analysis, we reestimated ED and inpatient cost per record, HCFRs, and NDRs when including records of undetermined intent alongside assaults to examine whether potential misclassification of assaults would affect results.37 To understand whether the exclusion of sports equipment (code Y08.0) affected NDR and HCFR estimates, we compared ED and inpatient cost per record with and without their inclusion.We used the underlying cause of death data from the Centers for Disease Control and Prevention Wide-ranging Online Data for Epidemiologic Research (WONDER) databaseAll analyses using HCUP data applied survey weights that cover more than 143 million ED and 35 million inpatient records each year and were conducted using Stata, version 16 (StataCorp LLC). The threshold of significance was set at α = .05 using 2-sided testing.38From 2016 to 2018 data, 2.8 million ED and 197 320 inpatient records were documented as assaults involving bodily force, blunt objects, sharp objects, or firearms only. This number was reduced to 2.7 million ED records when examining HCFRs and 2.4 million ED and 184 040 inpatient records when examining costs  had experienced bodily force; 13% (NEDS) and 11% (NIS) had been assaulted with firearms. Individuals with assault due to firearms had the lowest mean age  with most covered by Medicaid  or having no insurance . Across all assault mechanisms, most occurred in the Southern part of the US. The most common sites of injuries due to firearm assaults were the extremities  and multiple locations . In the inpatient setting, most bodily force (48%) and blunt object (41%) assaults involved patients who were White, and most sharp object (37%) and firearm (60%) assaults involved patients who were Black. Firearm assaults had the lowest ratio of ED to inpatient records (2:1); for every firearm assault recorded in the inpatient setting, there were 2 ED records, and bodily force had the highest ratio of ED to inpatient records (22:1).The A similar pattern was evident for NDRs and HCFRs; NDRs per 100 000 were 0.04  for bodily force, 0.03  for blunt object, 0.54  for sharp object, and 4.4  for firearm assaults. The HCFRs were 0.01%  for bodily force, 0.05%  for blunt object, 1.05%  for sharp object, and 15.3%  for firearm assaults. A crude examination of the In regression analyses after adjusting for confounders and using a complete-case analysis, ED costs for assaults involving firearms were higher by 99%  vs those for bodily force, 73%  vs blunt object, and 59%  vs sharp object assaults , and firearms contributed the most to inpatient costs ($1.6 billion). Combined ED and inpatient costs were highest for bodily force assaults ($2.6 billion), although this level was attained because bodily force assaults were more prevalent. Firearm assaults had the highest ED and inpatient costs per record, NDR, and HCFR, which were followed by sharp objects, and blunt force assaults (blunt object or bodily force) had the lowest. The highest total inpatient cost and inpatient cost per record for firearm assaults may be explained, in part, by the lower ratio of ED to inpatient records for firearms (2:1) compared with other mechanisms.28 Emergency department records for firearm assaults involved younger patients, on average, and fewer females than nonfirearm assaults. The same pattern was observed for inpatient records in addition to Black patients accounting for the most firearm assaults. The receipt of government insurance (Medicaid) by most individuals involved in assaults highlights the substantial cost borne by the public during initial hospital encounters and the socioeconomic context in which exposure to violence occurs; in particular, exposure to firearm violence for young male individuals who are Black33 or those in lower socioeconomic groups.39 As with Corso et al,11 we noted that greater total health care costs were associated with blunt force injuries  given their relative abundance. Although a variety of policies may serve to reduce violence in society, measures aimed at firearm violence may highlight a more cost-effective option for policies aiming to reduce morbidity and health care costs per incident.40Consistent with previous research, most violent injuries recorded in the ED involved males, individuals with public insurance or no insurance, and were recorded in hospitals in the Southern US.18 Our estimates are similar , albeit with differing time periods and data extraction . Miller and Cohen22 estimated the direct medical costs to be $27 299 for a gunshot wound and $16 178 for a stab wound. Although these estimates are older than our data and include costs beyond the initial hospital encounter, the differences are similar to those presented herein for inpatient costs . Evidence elsewhere12 suggests that the initial health care cost following a firearm injury may be a small fraction of the total economic cost and that the gap in costs between firearm and sharp object injuries may widen when considering longer-term productivity losses and mental health care costs.22 The younger average age of individuals who experience firearm assaults and their greater likelihood to die helps to explain this widening economic cost, for example, through years of life lost, let alone the humanistic toll on families and communities.41Emergency department and inpatient cost per record, NDRs and HCFRs were significantly higher when an assault involved a firearm. Using 2016-2017 HCUP data, the Government Accountability Office estimated the cost of initial treatment for a firearm injury to be $1478 for the ED and $30 703 for inpatient care.4 We found that assaults involving firearms were more likely to injure multiple locations as well as extremities. In regression analysis, wound location was significantly associated with cost, which may explain the widening cost differential between firearm and nonfirearm assaults after adjusting for confounders. Wound location may also relate to offender intent, ie, if the offender intends to inflict greater damage they may target multiple or specific body parts. Braga and Cook21 attempted to control for assailant intent by adjusting for injury location and number of injuries and found that gun caliber was significant in estimating fatality in a firearm assault. This result supports the argument that mechanisms may influence proximal victim outcomes in an assault.In addition to the characteristics of patients involved in assaults, differences in injury characteristics help explain the differences in costs across mechanisms. Bullets may carry considerably more force than other mechanisms and can cause greater damage to local and surrounding structures while also potentially injuring more locations on the body.17 estimate of 25%; although these authors examined all settings, for consistency in comparing across mechanisms, we restricted our analysis to the hospital setting. A comparison of HCFRs in the Figure suggests that patients are 15 to 1500 times more likely to die in the hospital due to a firearm assault compared with a nonfirearm assault. Cook,42 using ED data, found that individuals who were shot were more than 7 times as likely to die compared with those injured in a knife attack. Our estimate is twice as large as Cook’s estimate because he focused on individuals who were seriously injured in a knife attack. Our results are generally higher than earlier estimates of an individual’s death by a gun (0.41%), knife (0.13%), blunt instrument (0.04%), and unarmed (0.02%) robbery,43 although these estimates were older and relate only to robberies and so may not be directly comparable. Our results help to explain the near equivalent reduction in total homicides from policies targeting a reduction in firearm homicides even though substitution may occur in up to 30% of cases44 . Thus, policies targeting a reduction in firearm violence may reduce mortality and, to a lesser extent, health care costs even when substitution with other types of violence occurs. However, as we have shown, the differences in costs between firearm and nonfirearm assaults are still substantial, with firearms being associated with 59% to 118% higher costs.Emergency department and inpatient costs were 59% to 118% higher for firearm vs nonfirearm assaults after adjusting for confounders. We observed an even greater differential between NDRs and HCFRs for firearm compared with nonfirearm assaults. Our estimate of the HCFR for firearms was 15.3% which was lower than the Kaufman et al45 and it is estimated that a strict regime  would reduce firearm homicides by approximately 14% in the US over a 6-year period with an equivalent reduction in total homicides. Assuming a pro rata reduction in firearm assaults, a basic calculation suggests that, even when substitution occurs (approximately 30% of instances) toward the next most costly mechanism in our results (sharp objects), this strict regime may have been associated with a reduction in total combined ED and inpatients costs of approximately $247 million from 2016 to 2018: 14% of the total ED and inpatient costs ($1.7 billion) from initial hospital encounters due to firearm assaults during the same period (calculations in eTable 5 in the 40 especially for individuals of lower socioeconomic status and those of Black race. However, political partisanship may slow progress even when there is broad support across party lines for gun control policies at the mass level.46Stand your ground, right-to-carry, and child access prevention laws appear to be associated with the incidence of firearm deaths37 for example, injuries coded as unintentional or undetermined intent may actually be assaults. It is not possible to know which of these injuries should have been coded as assaults; however, our estimates of ED and inpatient cost per record and HCFRs were similar when reclassifying injuries of undetermined intent as assaults. There may be other classification issues that were beyond the scope of this research, for example, readmissions for an injury may be coded as an initial visit, which would overestimate the count of initial ED encounters. Furthermore, the HCUP includes only costs billed by the facility and not, for example, professional fees,47 so our results are likely an underestimation of the initial cost of hospital care. Although the counts of HCUP admissions were nationally representative for inpatients costs and HCFRs, missing ED costs meant that these estimates were not nationally representative, although the results did not differ substantially when imputing ED costs.This study has limitations. With the use of HCUP data to estimate hospital costs and HCFRs according to mechanism, misclassification bias may occur,From 2016 to 2018, although firearm assaults accounted for the fewest ED records relative to sharp objects, blunt objects, and bodily force, firearm assaults had the highest ED and inpatient costs per record, NDRs, and HCFRs. The findings of this study suggest that estimates of the total economic cost of firearm violence are useful in drawing attention to this public health issue; however, policies aiming at reducing the cost of firearm assaults should consider violence more broadly to understand the extent to which such firearm assault costs can be avoided.


DNA Virus and Host Plant Interactions from Antagonism to Endogenization” investigate the complexity of parasitic, mutual, or commensal virus interactions with the host cell and highlights the evolved diversity of DNA viruses infecting major crop plants, ornamentals as well as weeds.The findings and reviews in the article collection of the Research Topic on “Caulimoviridae, neither of them relying on chromosomal integration in their replication cycle DNA genomes in their capsids or to the family Caulimoviridae that encapsidate double stranded (ds)DNA genomes. Viruses being acellular, parasitic entities occur generally as episomes in their host cell on which their replication depends . Additionally, for geminiviruses the multifunctional replication initiator protein (Rep) encoded by ORF AC1 comprising also endonuclease activity is likely to play a key role in promoting host genome accessions of viral sequences , namely V2, C2, C4, and βC1 are developed to overcome plant defense mechanism and establish a sustainable infection. Furthermore, the authors reveal distinct functions of the investigated viral suppressors according to their subcellular localization, interactions and roles in symptom induction and intercellular movement.In order to infect their hosts successfully, viruses have evolved various approaches to overcome the RNAi based plant surveillance system in the nucleus as well as in the cytoplasm as illustrated and discussed by Majumdar et al.). The observed cross-kingdom RNAi, e.g., plant derived miRNAs targeting the viral capsid protein supports the hypothesis of a chimeric scenario for the origin of viruses, which postulates that replicons existed at an precellular stage and proteins for virion formation derived from the host . This is especially true for collections of germplasm from major food crops like yam and banana. The optimized multiplex PCR-dependent denaturing gradient gel electrophoresis facilitated the screening significantly and allowed comprehensive detection and analysis of endogenous Dioscorea bacilliform viruses . Umber et al. revealed the dynamics of activation for infectious endogenous pararetroviruses (EPRVs) in banana. The authors paid special attention in their risk studies to cultivation methods comparing tissue culture with field cultivation and indicated the impact of time and altitude respectively for activation on EPRVs in banana.The assembled publications of this Research Topic give examples for the dynamics in autonomous DNA virus evolution as well as in co-evolution with their hosts during endogenization. Thus generated viral diversity and spectrum of virus-host interferences require adapted detection methodology as well as risk assessments . Such comprehensive approach identified two previously unreported endogenous badnaviruses in the genus Solanum. In depth analysis of the genomic location of these endogenous badnaviruses found them adjacent or within the late blight resistance gene of their host Solanum melongena. Moreover, the authors located Ty-1 copia mobile elements–also known as Pseudoviridae- in this genomic niche. Future studies addressing the mechanisms that resulted in the co-localization of reverse transcribing elements such as Caulimoviridae and phylogenetically closely related Metaviridae as well as Pseudoviridae are highly desirable.Design of bioinformatics pipelines are seminal for exploration and functional analyses of integrated viral DNA sequences and related transcriptomes in the plant hosts .The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


The results are discussed in terms of their historical meaning and illustrate the exploitation of sources of dolomitic marbles other than the Greek Thasos source.The study of the provenance of dolomitic marble artefacts has become relevant since it was discovered that quarries of this marble other than that of Cape-Vathy located on the island of Thasos have been exploited since Antiquity. To improve our knowledge about the provenance of materials and the extent of their dispersion, multiple archaeometric studies were performed in the past including isotope analyses, petrography, cathodoluminescence, and elemental analyses. In the present work, solid-state nuclear magnetic resonance (NMR) spectroscopy has been added to this panel of techniques. NMR allows the characterization of the material at a molecular level by looking at different nuclei: carbon, magnesium, and calcium. Statistical analysis of the data collected on both quarry samples and archaeologic items was also implemented and clearly demonstrates the efficiency of a holistic approach for provenance elucidation. Finally, the first  During his escapades in the Tyrolean mountains, he also discovered limestone with the same physical properties which was named “Dolomiti” in honor of Dolomieu by his colleague Theodore de Saussure [The characteristics of dolomitic marbles have been described since 1791 by the Commander Déodat de Dolomieu, a French geologist, in a letter addressed to Mr. Picot de La Peyrouse and published in the Journal de Physique [Saussure .Since that time, the two varieties of white marbles, dolomitic and calcitic, have been studied and can be distinguished by their chemical composition: calcite is a crystalized calcium carbonate, whereas dolomite is a crystalized calcium magnesium carbonate. The two marbles were widely used in the ancient world, particularly during the imperial age in architecture and plastic arts, and the dolomitic marble was specifically chosen for fine arts because of its high quality and its relative hardness compared with calcitic marbles.Still today, many archaeologists attribute the provenance of an ancient artifact made of a coarse grain and a very uniform white dolomitic marble to the Cape-Vathy quarry of the Thasos Island ,4,5,6. WTwo strategies may be employed to elucidate marble provenance. The most commonly used strategy consists of comparing scientific data acquired for an archaeological item to those obtained from different quarries by different teams and reported in scientific publications. This strategy, which involves the elimination of certain sources at each stage, can be called a “phase-out” strategy. Two major methods are employed for this comparison: (i) the mineralogical and petrographic analysis of thin sections and (ii) the analysis of the stable carbon and oxygen isotope ratio. All the reference databases have been published for most of quarries known to be exploited during Antiquity: Greece, Italy , Spain , PortugaThe second strategy processes all the data together through principal component analysis to determine the marble source. For this purpose, it is necessary to collect all the measures on the same set of samples (quarries and artefacts). Therefore, to test this holistic approach, other techniques may be added to the panel of the complementary techniques usually used, without necessarily having a database on the techniques developed over several decades.13C NMR has been successfully developed for determining the provenance of some white calcitic marbles at a region scale [25Mg isotope) and calcium (43Ca isotope)—are investigated in order to track differences at the molecular scale between the dolomitic varieties that can be useful for their discrimination.This is this global approach that we decided to test in the present work, by collecting data on both geologic and archaeologic samples. To the established methods used, such as isotopic and elemental analysis, we add a third technique, solid-state nuclear magnetic resonance (NMR), which allows the deciphering of materials at a molecular level. In preliminary works, on scale  or even on scale . In the Finally, all the data collected were processed using statistical analysis—principal component analysis—and access was provided to the provenance of the dolomitic marble of some decorative architectural materials of the Roman Theatre of Zaragoza, as well as to some sculptural artefacts conserved in the archaeological Museum of Málaga.A large geological collection of dolomitic marbles related to the artifacts in this study was established. The collection includes dolomitic marbles from the Cape-Vathy quarry of the Thasos Island (seven samples provided by T. Kozelj ); the soIn order to test the experimental approach, five archaeological items were studied. Three of them  were sampled from the Archaeological Museum of Málaga, where they are currently conserved. They were discovered in different places in the vicinity of Málaga and they have already been the subject of a provenance study . All of 13C, 43Ca, and 25Mg solid-state NMR, whose physico-chemical characteristics are reported in 13C NMR spectra of all of the samples were recorded with a 9.4 T Avance NMR apparatus from Bruker  equipped with a broad band solid-state CPMAS 4 mm probe containing up to 200 mg of marble powder (CESAMO facilities) using the experimental protocol previously described [escribed .43Ca NMR spectra of one sample of each quarry and two archaeological items (29 MM and BL) were recorded with a 21.1 T Avance Neo  equipped with a 7 mm VTN HX low gamma probe whose rotor contains up to 600 mg of marble powder (IMEC-ISB-UCCS Lille facilities) using the following parameters, as described in [ribed in : spectra25Mg NMR spectra of one sample of each quarry and two archaeological items (29 MM and BL) were recorded on a 18.8 T Avance Neo  equipped with a 4 mm HX low gamma probe [ma probe  using th13C line shapes were simulated with Bruker Biospin software . 43Ca line shapes were accounted for assuming a central-transition second-order quadrupolar broadening defined by a quadrupolar coupling constant CQ and an asymmetry parameter ηQ, on top of the position  and a convolution by a Gaussian broadening rendering a distribution of δiso using DMFIT software [25Mg spectra simulations were performed using an “extended Czjzek” model [iso, distribution of isotropic chemical shift δiso, average quadrupolar coupling constant CQ, average asymmetry parameter ηQ, distribution of quadrupolar coupling constant ΔCQ, and “disorder weight” ε. For both quadrupolar nuclei, infinite spinning speed regime and perfect magic angle were assumed. Spinning sidebands of the external transitions  are not taken into account. The spectra were simulated as follows: software . 25Mg spk” model ,28. Apar3 68%, 3 mL of HCl 38%, 3 mL of HF 40%, and 1 mL of HClO4 72% . The mixtures were heated for 5 h at 130 °C in a closed container, and then at 190 °C until the total evaporation of all of the acids, as previously described [3 solution before the ICP-OES analysis  facilities). Ten discriminant elements with respect to provenance were dosed—Al, B, Ba, Fe, K, Mg, Mn, Si, Sr, and Ti, as previously described [Approximately 100 mg of each of the quarry and archaeological samples was dissolved in a 10 mL solution containing 3 mL HNOescribed .All collected data were analyzed with R version 4.2.0   and the 13C NMR, elemental analyses, and 13C/18O isotopic ratio (the ratio 13C/18O of most of the samples (19) studied in this work were already reported in previous works [43Ca and 25Mg NMR methods were tested to evaluate their potential for solving marbles’ provenance questions. The results are presented with respect to different purposes: developing analytical methods that are complementary to those that already exist, optimizing a confident provenance attribution strategy, and answering questions about dolomitic marble trade during Late Antiquity.The main goal of this study is to determine the provenance of marbles used to sculpt the five different artefacts displayed in samples 1 studied us works ). All of13C and 25Mg) or 600 mg (for 43Ca) of powdered marble samples; the quantity depends on the size of the NMR sample holder used for the experiments. However, because NMR is a non-destructive method, the same powder used for all the NMR experiments was subsequently used for ICP-OES (100 mg) and isotopic analyses (maximum 30 mg).First of all, it is important to note some peculiar specificity of the NMR experiments. Spectra were recorded on approximately 200 mg  and to the substitution of Ca or Mg by another element, leading to a broadening of the signal resonance [13C NMR chemical shifts of dolomite (170.8 ± 0.1 ppm) are slightly different than that of calcite (170.5 ± 0.1 ppm), the resolution limit is reached when the stone is composed of both phases. In this case, only one large resonance can be observed  can be partially used as provenance markers. 1/2—depending on the geographic location of the marble. 1/2 for all data; this highlights the limit of the location determination using only 13C NMR data. Indeed, strong overlaps prevent the provenance identification of most artifacts, except for 29 MM, probably made of Coín marble, confirming the previous identification [esonance . These sesonance ,38. In tobserved A. Noneth43Ca NMR spectra were successfully recorded despite the difficulties due to the low natural abundance of the isotope sensitive to NMR  and the best fit for simulating its shape was obtained with a Gaussian–Lorentzian ratio of 200/80, a quadrupolar coupling constant CQ of 1.4 MHz, and an asymmetry parameter ηQ of 0.1, values in agreement with previous reported results [iso 14.0 ppm) and the best fit for simulating its shape was obtained with a CQ value of 1.8 MHz and a ηQ value around 0.4. The 43Ca NMR chemical shift value observed for the dolomitic marble is close to the one previously observed for Mg-calcite [iso, CQ, and ηQ are reported for 43Ca spectra of dolomite. isotope ). 43Ca c 275 ppm ) allows  results ; dolomit-calcite , but to As NMR spectra were recorded in quantitative mode , the ar43Ca spectrum required a 24 h acquisition time, we chose to record three 43Ca NMR spectra of dolomitic marbles representative of each of the quarries, except for Mijas, and two artifacts, one from the Roman Theatre of Zaragoza (BL) and one from the museum of Málaga (29 MM). They are displayed in Q around 1.7 MHz, ηQ between 0 and 0.3 and a LB/GB ratio of approximately 40/40; see table inserted in While each 25Mg NMR spectra were also recorded for the same five samples as those analysed for 43Ca NMR (three quarry samples and two artifacts): this isotope has a 5/2 spin and a magnetogyric ratio (−1639 × 107 rad s−1T−1) lower than 43Ca. Nevertheless, its 10% natural abundance makes it easier to observe than calcium /Mg(2) 31:64 for 29 MM, similar to the proportion observed for CN1), as viewed by 13C NMR, and confirming once again its provenance proposed by Lapuente et al. [25Mg spectrum simulation reveals proportions between the two Mg sites close to those of the Cape-Vathy quarry . These first results are very encouraging and will require the analysis of more quarry samples in the future. calcium . 25Mg NMied here . BecauseICP-OES was applied to quantify eight minor elements present in the samples: Al, B, Ba, Fe, K, Mn, Si, Sr, and Ti, as well as one major element, Mg. These elements were chosen because they have been shown to discriminate between geological locations . The Mg 13C versus d 18O isotopic data collected in different publications for the two Malaguese quarries Coín and Mijas [18O < −4 PDB (Pee Dee Belemnite)), whereas the Spanish marbles are concentrated in its right part (δ 18O > −4 PDB), but without a differentiation between the two quarries of Coín and Mijas; the Thasian dolomitic marble straddles these two groups of values . This illustrates the sensitivity of NMR spectra to the presence of paramagnetic elements. PCA results allow the identification of the provenance of two of the items: 6 MM from Coin and 26 MM from Mijas, as previously suggested by Lapuente et al. [All methodologies taken independently cannot provide provenance proof for most of the artifacts studied. As an additional example, we report in nd Mijas  and fromnd Mijas , as wellnd Mijas . Three dlues see . When thNMR data B. The fi13C NMR, and isotopes). The first two axes of the PCA explain 71%  of the observed variance. Mg content was the main contributor to the construction of the first axis (20%), while iron content, NMR intensity, and Δν1/2 were the main contributors to the construction of the second axis . Interestingly, the separation between Thasian and Pyrenean marbles is clearly achieved and allows for the identification of the provenance of the marble used to make the decorations of the Roman Theatre of Zaragoza. The PCA suggests that BL is from Thasos and not from the Pyrenees, in accordance with the preliminary results obtained with 25Mg NMR.Thus, PCA was performed by adding isotopic data to compositional and NMR data C. PCA waThe main archaeological goal of this work was to shed light upon dolomitic marble trade during Antiquity, and especially the exportation of Thasos dolomitic marble in all of the Mediterranean Basin that persisted for more than a millennium (6th century B.C. to 6th century A.D. ), versusFor this purpose, the provenances of the marble used to sculpt different artifacts were identified: some decorations of the prestigious Roman Theatre of the antique town of Caesar Augusta  and statues discovered in the region of Málaga. All of these artifacts date from the 1st to 2nd centuries A.D. and the strategy developed is based on a comprehensive approach that consists of processing a set of analytical data composed of judiciously chosen geologic marbles with respect to the artifacts studied and the artifacts themselves. This strategy allows to add original scientific methods on a obtainable series of samples.13C, 43Ca, and 25Mg. Each of them provides complementary information able to help in the decision-making process. 13C signal is sensitive to the presence of paramagnetic ions in its vicinity such as iron, manganese, and so on. 43Ca NMR spectrum allows to distinguish and quantify dolomite and calcite crystals and 25Mg is sensitive to structural disorder, i.e., imperfections in the crystalline lattice, that necessarily occurs because of the non-stoichiometric nature of dolomite rocks. The latter nucleus presents an interesting performance for provenance discrimination owing to its ability to quantify the two distinct Mg environment of dolomitic marbles.NMR has been selected because of its non-destructive property and its capability to obtain information on the atomic-scale structural environment of different nuclei. Three nuclei were tracked in this study, carbon, calcium, and magnesium, through their respective NMR-sensitive isotope A common problem in the determination of the provenance of marbles is the fact that the analytical data collected by most authors have been applied to different collections of samples, hindering a global multivariate analysis. To date, no single technique is able to determine the provenance of a marble. To overcome these difficulties, it is recommended, first, to use different and complementary methods in terms of collected information  and, second, to acquire all the data on the same collection of samples in order to proceed to the statistical data analysis for determining reliable provenance. This is why we collected NMR and ICP-OES data on samples already analyzed by petrography and cathodoluminescence by one of us, and whose isotopic ratios were already measured , and 29 MM, a male figure believed to be a distinguished person wearing a mantle, and found in Cartima, Málaga province dating from the first half of the 2nd century AD, are made of local dolomitic marble, and more particularly from the quarry of Coín. 24 MM, a fountain-sculpture from the villa de el Secretario, found at Fuengirola, Málaga province and dating from the mid-2nd century AD, is made of dolomitic marble extracted from the Mijas quarry. Were the two quarries exploited at different times, or were these quarries exploited for different purposes and/or clients depending on their social hierarchy? Regardless, in all these cases, the local marble source was preferred.The decoration of the Roman Theatre of Caesar Augusta, BL, has been identified as a Thasian marble. In this case, the farthest marble was favored: is it related to transport conditions—the sea route being easier than the land one? Or is it linked to the prestige of the building? We will let historians answer these questions!