Pueraria montana var. lobata), a plant native to Southeastern Asia, has become a major noxious weed covering millions of hectares in the Southern United States. A kudzu patch displaying virus-like symptoms located in Ackerman, northeastern Mississippi (MS), was used as a source for virus isolation and characterization involving mechanical and vector transmission, ultrastructural observation, surveys, Sanger and high-throughput genome sequencing, and sequence analyses. The results revealed the presence of a new potyvirus in infected kudzu, closely related to wisteria vein mosaic virus (WVMV) and provisionally named kudzu chlorotic ring blotch virus (KudCRBV). Genome features and pairwise comparison with six WVMV genomes currently available in GenBank and three additional isolates from MS sequenced in this work suggest that KudCRBV is likely a member of a new species in the genus Potyvirus. Furthermore, under experimental conditions, KudCRBV was successfully transmitted by cotton and potato aphids and mechanically to soybean and beans. A state-wide survey revealed several kudzu patches infected by the virus in northern MS.Kudzu ( Pueraria montana var. lobata (Willd) Maesen & Almeida) is a leguminous trailing/climbing perennial vine originating from Southeastern Asia. It was originally introduced to the United States during the Philadelphia Centennial Exposition in 1876 and was publicized as an easy-growing ornamental plant. Shortly afterwards, kudzu was presented at the New Orleans exposition in 1883, attracting the interest of homeowners as an ornamental plant for use in residential ornamentation and for shading of porches [Kudzu —kudzu can currently be found growing in Arizona, Oregon, and Washington too .Kudzu adapted exceptionally well to the climate of the Southern U.S. As a result, it continued to expand quickly, spreading to 31 states, and became a serious and costly problem. Current estimates suggest that kudzu approximately covers more than 3 million ha in the Southeastern U.S. and continues to expand at an approximate rate of 50,000 ha/year . Due to gton too , as wellSince kudzu has an aggressive and rapid growth pattern, it has the ability to outcompete vegetation nearby, from native grasses to fully mature trees, by covering them A and intPhakopsora pachyrhizi) [Megacopta cribaria), kudzu is also susceptible to a few viruses. Indeed, infections by tobacco ringspot virus  and soybean vein necrosis virus  have been reported from the Southern U.S. over the past decade [Crinivirus (family: Closteroviridae) and Emaravirus (family: Fimoviridae) and were observed in coinfections with KuMV.Kudzu is a host for several important plant pathogens and pests in the U.S. In addition to acting as an additional over-wintering host for the soybean rust fungus (hyrhizi)  and servt decade ,7,8. Curt decade . Howevert decade . Two of Potyvirus  are viruses with flexuous filamentous virions (~700–900 nm long and 11–13 nm wide) containing a genome made up of polyadenylated, positive-sense RNA generally 9.7–11 kb in size [In the current paper, we report a comprehensive characterization of a new virus with features typical of potyviruses and which is possibly a member of a new species. Members of the genus  in size .Wisteria sinensis) samples were used in various phases of the study (more details provided later in this section).The primary plant material used for this research was derived from a kudzu plant with the lab code “Ack01”, located in the town of Ackerman, Choctaw County, Mississippi. The plant material displayed virus-like symptoms in the form of chlorotic ring blotches and distinct line patterns B. AdditiNicotiana benthamiana were used as controls. The extracts were submitted to selective enzymatic digestions with RNAse-free DNase, DNAse-free RNAse in high-salt conditions , and Proteinase K prior to elimination of DNA and ssRNA traces. Purified dsRNAs were analyzed in 1% TAE agarose and visualized by staining with ethidium bromide. The approximate size of the dsRNA under investigation was estimated by comparison with replicative forms of isolates of grapevine leafroll-associated virus 2 [Double-stranded RNAs (dsRNAs) were isolated from 10 g of foliar tissue collected from the symptomatic sample “Ack01” using double phenol–chloroform extraction followed by CF-11 column chromatography . Foliar  virus 2 , bell pe virus 2 , and Jap virus 2 , routineSmall foliar tissue fragments, excised from the symptomatic foliage of “Ack01”, were used for observation of possible cytopathological alterations associated with the infection by the studied virus. Fragments were processed according to standard procedures , i.e., fIn this study, genome sequencing was performed for the kudzu virus as well as for three isolates of wisteria vein mosaic virus (WVMV) from Mississippi. Sequencing of the kudzu virus was originally performed via the Sanger methodology of random primer-generated cDNA clones starting from a dsRNA template as previously described ,19.Genome sequencing of the three WVMV isolates and verification of the kudzu virus genome were performed via Illumina-based high-throughput sequencing (2 × 150 nt pair-end reads) of ribo-depleted total RNA preparations performed on an Illumina HiSeq3000 platform on a custom basis at the Roy J Carver Biotechnology Center (University of Illinois at Urbana–Champaign). For that purpose, total RNA was extracted from 0.1 g of foliar tissue with a Spectrum TM Plant Total RNA kit , checked for quality/quantity with a Qubit 3.0 fluorometer , and shipped overnight on dry ice to the sequencing facility. The raw sequencing data  were initially checked for the quality, and high-quality reads were de novo assembled into larger contigs and further analyzed with the Geneious Prime v. 2022.0.1 software suite  and/or the OmicsBox v. 2.2.4 package .Sequences of the 5′ non-coding regions (NCRs) of virus genomes were completed using a SMARTer RACE 5′/3′kit (Takara Bio/Clontech). The identities of the virus sequences were determined by BLAST comparison (BLASTn and BLASTx) against the GenBank database in June/July 2022. A subset of WVMV complete genomes available on 1 December 2022 was downloaded to a local database and used for in-depth pairwise comparisons with the studied virus. Pairwise genetic identity calculations were estimated and color-coded distance matrices were generated by the Sequence Demarcation Tool (SDT) [Complete sequences of a kudzu virus were aligned with a set of potyviral genomes representative of the current taxonomy  using MATo better understand the incidence and distribution of this virus, we performed a survey with sampling throughout the entire state of Mississippi. A total of 103 samples, consisting of 30–35 cm long tips of the vines with young leaves, were collected over the two seasons (late May—early November 2018) from different locations and tested initially in DAS-ELISA using a “general potyvirus” monoclonal antibody kit . The samples that were determined to be virus-positive in DAS-ELISA were further tested in RT-PCR with the virus-specific primers (KudV-F 5′CTAAGCCAACTCTGAGACAAATCA3′ and KudV-R 5′GCGGTGGGCCCATGCCCA3′) designed to amplify a 349 nt long portion of the viral genome. The final identification was achieved by custom-based amplicon Sanger sequencing performed at the Eurofins-Operon facility in Louisville, KY, USA.An investigation into possible alternative hosts of the virus was carried out by collecting and testing tissue from several plants naturally growing in the same ecosystem  as the originally infected kudzu plant. To that purpose, we surveyed the area surrounding the plant “Ack01” for two consecutive years (2019–2020), with sampling performed in May and October of each year. During the survey period, a total of 66 plants belonging to nine different botanical species  were tesMacrosiphum euphorbiae (Thomas), and the cotton aphid, Aphis gossypii (Glover), were used in vector transmission trials. The potato aphids were collected from wild lettuce, Lactuca virosa L., in the area surrounding the original symptomatic kudzu plant, while cotton aphids were collected from bean (Phaseolus vulgaris L.) and cotton (Gossypium hirsutum L.) located a few km from the kudzu patch. Transmission experiments were carried out in 2017. Aphids were identified using dichotomous keys available in a reference book on aphids that impact crop plants [Two species of aphids collected in Mississippi, identified as the potato aphid, p plants .Glycine max) plants of the cultivar Asgrow 4903 (AG4903).Symptomatic kudzu leaf tissue from a “donor” plant Ack01 was placed in a Petri dish and aphids were transferred from natural hosts onto symptomatic tissue using a soft-bristled paint brush and left for two hours. During this period, the aphids were under intermittent observation to ensure that feeding occurred. After two hours, the aphids were transferred to caged kudzu plants with the accession nos. 9227 and 434246 grown using seed obtained from the National Seed Storage Laboratory and soybean  were used in this study for each of the two aphid species . A minimum of ten aphids were carefully placed directly on each individually caged recipient plant. The plants were briefly removed from cages after 24 h, sprayed with an insecticide to kill aphids, and maintained under controlled conditions with daily visual monitoring for symptom development. Soybean cultivar AG4903 was treated in the same manner.v/w), onto cellite-dusted leaves of soybean cv AG4903, tobacco (Nicotiana tabacum cv ‘Turkish’), bean (Phaseolus vulgaris cv ‘Bountiful’), squash (Cucurbita pepo cv ‘Zucchini grey’), two cultivars of cucumber , watermelon (Citrullus lanatus cvs ‘Crimson sweet’ and ‘Black diamond’), pea (Pisum sativum cvs ‘Zipper cream’ and ‘Pinkeye purplehull’), and quinoa (Chenopodium quinoa). Each type of plant was represented in 5 replications, except soybean, which was represented by 10 plants. The plants were maintained under a constant temperature regime (approximately 20–22 °C) in a greenhouse for 30 days post-inoculation (d.p.i). The plants were visually observed for symptom expression on a daily basis, starting from three days post-inoculation.Mechanical transmission to a range of young herbaceous plants was attempted by rubbing sap from symptomatic apical leaves collected from “Ack01” in 0.1 M phosphate buffer, pH 7.2 . In silico analyses suggested the polyprotein processing at the cleavage sites presented in A large, 3095-codon-long ORF covering approximately 96% of the whole genome codes for a polyprotein with an estimated In the initial BLAST alignments performed on complete nucleotide sequences in July 2020, KudCRBV shared the greatest level of identities  with the two near-complete sequences deposited in GenBank as wisteria vein mosaic virus isolates Ce-JH  and JEBUp . However, identity with a complete sequence of an original isolate of WVMV from Beijing   was founIn phylogenetic analyses performed on complete amino acid sequences of the polyproteins of reference potyviral isolates, the virus from kudzu was placed in a well-supported subclade composed of soybean mosaic virus (SMV), watermelon mosaic virus (WMV), and wisteria vein mosaic virus (WVMV) within the “bean common mosaic virus clade”  of potyviruses . Indeed,Potyvirus, prompted a state-wide survey of wisterias in MS that aimed to identify suitable sources and to generate molecular data on local isolates of this virus. As a result of that survey effort, three wisteria samples were determined to be positive for WVMV following potyvirus-specific DAS-ELISA. The resulting virus-positive plant material was submitted to HTS sequencing by Illumina.Thus, inconclusive results from initial comparisons of KudCRBV with a limited number of the WVMV genome sequences available at the time of the initial analyses (July 2020) regarding their relationships, as well as overall taxonomic position of a kudzu virus within the genus After raw sequence assembly and the performance of 5′RACE experiments for the first two isolates, their complete genome sizes differed by a single nucleotide (not counting the polyA tail): 9694 vs. 9693 nt for isolates MS14-19 and MS20-26. Additionally, a coding-complete sequence 9664 nt in size containing partial 5′UTR and entire coding and 3′UTR sequences was assembled only from HTS reads for the isolate MS12-11. Genomes of the three isolates coded for a major polyprotein with a molecular mass of ~354 kDa and a small PIPO protein 74 aa in size.Pairwise comparisons revealed that the WVMV population present in MS is rather conserved, as the genomes of the three isolates shared mutual nt identities ranging from 95.3% to 98.9%, depending upon the specific combination used for comparison. Putative polyproteins of the three WVMV isolates shared identical lengths of 3092 amino acids and had highly conserved contents, differing mutually by only 1% (MS14-19 vs. MS20-26) to 2.7% (MS14-19 vs. MS12-11). However, they shared only 76.4–76.6% nt sequences genome-wide with KudCRBV.Generation of data on genomes of the three local WVMV isolates, along with timely availability of sequences of two German sources of this virus from the DSMZ collection, allowed for a more robust analysis to understand the nature of KudCRBV-Ack01 relationships to allied viruses . Similar tree topologies were obtained with a richer dataset which included a few additional isolates from Italy, Iran, Australia, and two from China; KudCRBV and two WVMV isolates from Korea formed an evolutionarily distinct lineage from the Wisteria-infecting WVMV isolates.Phylogenetic analyses of either complete ORF (nt) or polyprotein (aa) sequences revealed the presence of two distinct clusters, one represented by KudCRBV and two WVMV isolates (Ce-JH and JEBUp) reported from non-wisteria hosts from Korea . KudCRBV was found to be most related to WVMV-JEBUp, an isolate reported from soybean .On the other hand, three WVMV isolates from MS were closely related to two recently reported German isolates, WVMV-PV1026 (GenBank OQ993365.1) and WVMV-PV1105 (GenBank OQ731912.1), with no currently associated publication, and an Iranian isolate WVMV-Ir (GenBank MN514947.1)  sharing To better understand how the genetic distances observed in analyses of KudCRBV and WVMV populations compare to those between populations of WMV and SMV, we included several isolates of those two viruses in the next round of analyses. KudCRBV shared similar levels of identity, around 73–74%, with SMV and WMV isolates. The highest level of mutual sequence identities was observed between SMV and WMV, varying between 78.8% and 79.2%, therefore significantly exceeding that between KudCRBV and WVMV isolates from wisteria . SimilarThe identity levels of ten KudCRBV mature proteins with those encoded by closely related viruses, deduced by in silico processing of the primary polyproteins, varied according to the protein and virus used for comparison . Of impoOnce the viral cause of the symptoms on the kudzu contained in the patch in Ackermann, MS, was identified, two KudCRBV-centered surveys were conducted. In the first survey, which aimed to investigate possible additional natural hosts of the virus, foliar tissue was collected from 66 plants belonging to nine different botanical species from the area immediately surrounding the infected kudzu and tested by RT-PCR for the virus. No plant sample other than kudzu was determined to be infected by KudCRBV.A state-wide survey of 103 kudzu samples collected from various locations revealed 5 positive samples in DAS-ELISA with “universal” potyvirus monoclonal antibodies. Additional RT-PCR tests, followed by sequencing of 349 bp long amplicons confirmed that all five samples were indeed infected by KudCRBV, as they shared high levels (97–98.5%) of identical nucleotide sequences. Curiously, all six  kudzu patches were in the northernmost part of the state A.Vector transmission trials were successful, although at variable rates. The cotton aphid transmitted the virus to soybean (two out of five plants) and kudzu accession no. 9227 , while no infections developed in kudzu no. 434246. On the other hand, the potato aphid successfully inoculated both kudzu genotypes, but did not transfer KudCRBV to any of the five soybean plants of the AG4903 cultivar used in these trials. The RT-PCR experiments showed transmission to two out of five plants of kudzu no. 9227 and only to a single plant in the case of kudzu no. 434246.In mechanical transmission attempts, KudCRBV caused systemic infections in 5 out of 20 soybean plants and 4 out of 12 cv ‘Bountiful’ bean plants. KudCRBV provoked systemic veinal discoloration/clearing, chlorosis, and mosaic in AG4903 soybean plants approximately two weeks post-inoculation B. In FrePotyvirus, for which the name kudzu chlorotic ring blotch virus (KudCRBV), based on the natural host and the originally observed symptoms, is proposed. The virus shares properties with previously characterized potyviruses, as it induced cytoplasmic structures within infected kudzu plant material that are typical of infections by this group of viruses and is aphid transmissible. Indeed, the virus has been successfully transmitted by the two aphid species used in the study under experimental conditions. Taking into consideration the relatively widespread occurrence of cotton and melon aphids in MS, we cannot help but suggest their role in the epidemiology of the virus in nature. Although the survey performed on plant samples belonging to nine different botanical species collected from the vicinity of the original kudzu patch in Ackerman, MS, USA, failed to identify additional natural hosts of this virus, transmission trials performed under controlled conditions also determined that KudCRBV can infect two additional leguminous hosts. Of particular significance is the fact that soybean, one of the major agricultural commodities in the state, was determined to be susceptible to KudCRBV infection using both an inoculation procedure with cotton aphids and by mechanically rubbing virus-infested sap onto soybean leaflets. Of additional noteworthy importance is the fact that kudzu has been reported to occur in 81 of the 82 counties in Mississippi [The virus discovered and characterized in the current report is herein described as a new member of the genus sissippi . Many ofIn addition to the original kudzu patch in Ackerman, the state-wide survey revealed several additional KudCRBV foci in the state. The five additionally infected kudzu plants were all collected in the northern part of the state (five infections in 49 samples collected north of Ackermann), while KudCRBV was not detected in any of the 54 samples collected from the central or southern part of Mississippi. It is not clear whether this distribution pattern is a result of the use of KudCRBV-infected material during a major planting campaign almost a century ago or of natural spread over time either by airborne vectors (aphids) or possibly via seed-transmission, with the possible involvement of birds as major vehicles for their transport over greater distances. Moreover, given that kudzu seed requires a level of scarification prior to germination, it is more likely that the movement of virus-infested seed by birds has occurred. However, further studies of possible pollen and/or seed transmission might shed light on the current virus distribution in Mississippi. It is likely that there are more virus-infected kudzu patches in Mississippi, since the invasive weed is very widespread throughout the state and only a fraction of its population has been sampled and tested in this work.Generally, the discovery of pathogenic viruses in an invasive and destructive plant such as kudzu might appear to be an exciting opportunity for their employment as part of an integrated control strategy. However, the use of exotic viruses, such as KudCRBV, as biological control agents could be highly controversial as it poses a risk for economically important crops.Although we have no experimental proof of natural infections of soybean by KudCRBV in MS at this time, our results clearly demonstrated the susceptibility of this crop to the virus. This fact should not be overlooked considering the economic importance of soybean production in MS and more broadly in the U.S. and possibly warrants an increased awareness regarding the potential future threat to soybean. There are several well-studied cases of virus “spillover” from their natural non-cultivated hosts (wild reservoirs) and adaptation to agricultural ecosystems/crops worldwide . It shoPotyvirus have genome sequences that are generally less than 76% identical in nt content, and their encoded polyproteins share less than 82% identical amino acids. The thresholds for species demarcation using nucleotide identity values for the individual coding regions range from <58% for P1 up to <80% aa identity for CP [According to currently valid molecular demarcation criteria, members of different species in the genus y for CP ,38,39. HKudCRBV likely originated in the Far East and was introduced in the U.S. via infected planting material during in the late 19th or early 20th century. Indeed, it is evolutionarily most closely related to WVMV and a few additional viruses within the “SMV cluster” of the “BCMV clade”, one of two of the largest lineages of potyviruses that apparently radiated in Southeast and East Asia approximately 3600 years before present (ybp) ,41,42. OPotyvirus, of Asian origin, known to affect plants belonging to several species in the genus Wisteria  causing Wisteria mosaic disease, a disease originally reported from the U.S. in the mid-20th century [Wisteria spp. in various places worldwide, including different European countries, including the United Kingdom [W. sinensis collected in MS. Fortuitously, the genome data of two German isolates of the virus from the DSMZ collection were made publicly available just in time for the preparation of this manuscript and were included in the analyses.WVMV is a member of the genus  century . However century . The vir Kingdom , Poland  Kingdom , and, mo Kingdom . The vir Kingdom , New Zea Kingdom , and on  Kingdom ,49,50, a Kingdom ,51,52. WPotyviridae Study Group (SG) [Uraria crinita in 2014, which was found to be significantly different from the sequences of another 304 isolates used in that comprehensive study [Phylogenetic analyses and pairwise analyses performed in the current work, independent of the type of dataset used , revealed a clear-cut divergence of known WVMV populations into two distinct lineages  , suggestve study  and suffPotyvirus, suggesting that these three viruses belong to the same taxon, it is questionable whether they should be grouped together with the rest of the WVMV isolates, as they clearly belong to an evolutionary distinct lineage. In phylogenetic trees, distances between the two WVMV clades are comparable to that between SMV and WMV, which are classified as two different species (Potyvirus.While the identities of KudCRBV with two genomes deposited as isolates of WVMV significantly exceed the proposed species demarcation criteria for the genus  species . Moreove species . FurtherW. sinensis) independently of their geographic origin  collected in the Southern U.S., whereas the other two were characterized from two distinct hosts in Korea, WVMV-Ce-JH from jack bean  and WVMV-JEBUp from soybean. Therefore, this group of viruses have a rather distinct gamma of hosts compared to those from “Clade B”  or it could be “lumped” together with all the WVMV isolates as a distinct strain adapted to a particular host (kudzu). Based on the extensive results from this study, in particular an in-depth comparison of genetic divergences between populations of closely related viruses , which are classified as different species, our opinion is that the process of speciation in the case of KudCRBV (and the other two WVMV isolates) has been sufficiently completed to warrant the creation of a new and distinct taxon in the genus


LAS), narasin (NAR), or virginiamycin (VRM) on rumen fermentation parameters, apparent nutrient digestibility, and blood parameters (Exp. 1), as well as feed intake and performance (Exp. 2) of Nellore cattle consuming a forage-based diet. In Exp. 1, 32 rumen-fistulated Nellore steers  were assigned to a randomized complete block design. Within block, animals were randomly assigned to one of four treatments: 1) forage-based diet without feed additives (CON), 2) CON diet plus 13 mg/kg of dry matter (DM) of NAR, 3) CON diet plus 20 mg/kg of DM of sodium LAS, or 4) CON diet plus 20 mg/kg of DM of VRM. No treatment effects were detected (P ≥ 0.32) for intake and apparent digestibility of nutrients. Steers fed NAR had the lowest (P ≤ 0.01) molar proportion of acetate on day 28, 56, and 112 vs. CON, LAS, and VRM steers, whereas acetate did not differ (P ≥ 0.25) between LAS, VRM, and CON steers from day 28 to 84. On day 112, steers fed LAS had a lower (P < 0.02) molar proportion of acetate vs. VRM and CON, whereas it did not differ between CON and VRM (P > 0.33). Steers receiving NAR had a greater (P ≤ 0.04) ruminal propionate vs. CON, LAS, and VRM, whereas LAS steers had greater (P < 0.04) propionate vs. CON and VRM steers on day 28 and 112, and it did not differ (P > 0.22) between CON and VRM. In Exp. 2, 160 Nellore bulls were blocked by initial shrunk BW (212 ± 3.1 kg) in a 140-d feedlot trial. Diets contained the same treatments used in Exp. 1. Bulls fed NAR had greater (P < 0.02) average daily gain (ADG) vs. CON and VRM, and similar (P = 0.17) ADG between NAR and LAS, whereas ADG did not differ (P > 0.28) between LAS, VRM, and CON bulls. A treatment effect was detected (P = 0.03) for dry matter intake, being greater in NAR vs. CON, LAS, and VRM bulls, and similar (P > 0.48) between CON, LAS, and VRM bulls. A tendency was detected (P = 0.09) for feed efficiency, which was greater (P < 0.02) in NAR bulls vs. CON and VRM, and similar (P = 0.36) between NAR and LAS bulls. From day 112 to 140, bulls receiving NAR were heavier (P < 0.03) vs. CON, LAS, and VRM bulls, but no differences were observed (P > 0.51) between CON, LAS, and VRM bulls. Collectively, ruminal fermentation profile and intake were impacted by NAR supplementation, which partially contributed to the enhanced performance of Nellore bulls receiving a forage-based diet.Two experiments were designed to evaluate the impacts of supplementing lasalocid ( Supplementing narasin to beef cattle for 140 d might be a feasible nutrition tool to optimize rumen fermentation characteristics and productivity in grazing cattle systems. Bos indicus Nellore cattle fed forage-based diets. Additionally, there is a significant gap to be filled by research involving molecules capable of altering the fermentation process and, consequently, performance of Nellore cattle fed forage-based diets  are commercially available and commonly fed in many phases of beef production systems. Although their ruminal mechanisms are similar, performance might vary depending on dosage, animal, and diet  is a nonionophore produced by a specific strain of Streptomyces virginiae , as well as feed intake and growth performance (Exp. 2) of B. indicus Nellore cattle consuming forage-based diet.Thus, we hypothesized that supplementing or not LAS, NAR, or VRM would affect nutrient digestibility, change rumen fermentation parameters, and increase productivity of This study was conducted at the University of São Paulo, Piracicaba campus . Experimental procedures involving animals were reviewed and approved by the Ethics Committee on Use of Animals of School of Veterinary Medicine and Animal Science . The experimental design, measurements, and analyses used herein are similar described by B. indicus) steers  were assigned to individual pens  in a randomized complete block design (n = 8), according to their initial shrunk BW. Within block (n = 8), animals were randomly assigned to one of four treatments: 1) forage-based diet without feed additives , 2) CON diet plus 13 mg/kg of dry matter (DM) of NAR , 3) CON diet plus 20 mg/kg of DM of sodium LAS , or 4) CON diet plus 20 mg/kg of DM of VRM . The administration rates of NAR, LAS, and VRM used herein were according to the manufacturer’s recommendation. The experimental period lasted 140 d and was divided into five periods of 28 d each  as in Thirty-two rumen-fistulated Nellore (Cynodon dactylon (L.) Pers] and 1% of concentrate , which was used as a delivery vehicle for the additives (DMI). For instance, for steers consuming 5.0 kg of forage, the concentrate would contain 65, 100, and 100 mg/kg of DM of NAR, LAS, and VRM for NAR, LAS, and VRM, respectively. Steers from the CON group also received the same concentrate without including feed additives. From day 0 to 140, animals were individually fed the feed additive treatments once daily (0800 h) prior to haylage feeding to avoid a small amount of concentrate being mixed with hay and compromising intake of the feed additive treatments  was obtained by multiplying the total N content by 6.25. Calculation of haylage and concentrate total digestible nutrients, net energy for maintenance and gain were performed according to Samples of feed, orts, and feces were dried in a forced-air oven at 60 °C ; #930.15Individual shrunk BW was collected on day 0 after 14 h of feed and water withdrawal and used to calculate initial BW and to perform the randomization into blocks and treatments. Forage, concentrate, and total DMI were recorded daily from each pen by collecting and weighing nonconsumed feed (forage only). Samples of the offered and nonconsumed feed were collected daily from each pen and dried for 24 h at 105 ± 2 °C in forced-air ovens for dry matter calculation.SCFA; acetate, propionate, butyrate, isobutyrate, valerate, isovalerate), as well as the acetate:propionate (Ac:Pr) ratio, and total SCFA. Frozen ruminal samples were prepared for analysis by thawing, centrifuging  for 10 min at room temperature and analyzed for SCFA and rumen ammonia according to procedures described by On day 0 , 28, 56, 84, 112, and 140 of the experimental period at 0, 6, and 12 h after concentrate feeding, ruminal fluid samples were collected (approximately 100 mL per sample time) by squeezing the ruminal contents into four layers of cheesecloth and the ruminal fluid pH was immediately determined . Approximately 50 mL of ruminal fluid were collected, pooled across all sampling times , within each experimental period, and stored (−20 °C) for subsequent analysis of rumen ammonia and molar proportions of individual short-chain fatty acids . All blood samples were immediately placed on ice, subsequently centrifuged  for plasma collection into 1.5 mL tubes , and stored at −20 °C until analysis. The blood parameters were determined in the Automatic Biochemistry System—Model SBA-200 . Commercial enzymatic kits from Labtest Diagnostic SA  were used for the determination of plasma concentration of glucose and urea. All samples were analyzed for ruminal ammonia, glucose, and urea concentration within a single assay, and the intra-assay CV was 4.91%, 1.78%, and 4.44%, respectively.Blood samples were collected on day 0 , 28, 56, 84, 112, and 140 of the experimental period at 6 h after feeding, via coccygeal venipuncture into commercial vacutainer collection tubes with glycolytic inhibitor and anticoagulant KB. indicus Nellore yearling bulls were assigned to pens in a randomized complete block design (n = 10), according to their initial shrunk BW . The experimental period lasted 140 d, divided into five periods of 28 d each. Bulls were kept in a covered feedlot  with a concrete floor, feed and mineral bunk, and waterer. Within blocks (n = 10), animals were randomly assigned to the same treatments as in Exp. 1.One hundred and sixty C. dactylon (L.) Pers] and 4% of concentrate (composed of 50% ground corn and 50% ground citrus pulp dry), used as a delivery vehicle for the additives. Haylage was chopped using a vertical mixer  for about 15 min, which resulted in an average particle length distribution of 60.1% ± 1.8% > 19 mm; 23.0% ± 1.0% > 8 mm; 8.9% ± 1.7% > 4 mm; and 8.0% ± 1.3% on bottom sieve according to Penn State Particle Separator procedures  and gain:feed (G:F) efficiency, bulls were individually weighed on day 0, 28, 56, 84, 112, and 140  after 14 h of feed and water restriction. DMI was evaluated daily from each pen within each period by collecting and weighing nonconsumed haylage weekly. Haylage and total DMI of each pen were divided by the number of bulls within each pen and expressed as kg per bull/day. Total BW gain and DMI of each period were used for bull G:F calculation. Samples of feed and orts were collected weekly, pooled across all weeks within each period, and analyzed for nutrient profile as aforementioned for Exp. 1.To calculate average daily gain  or pen (Exp. 2) as the experimental unit and the MIXED procedure of SAS , with Satterthwaite approximation to determine the denominator df for the test of fixed effects, and animal(treatment) as random variable for Exp. 1. In Exp. 2, however, pen(treatment) and animal(pen × treatment) were used as random variables for all variables, except for DMI and G:F that used pen(treatment) as random variables. Model statements contained the effects of treatment, period or day, treatment × day or period interaction, and block as an independent variable. The specified term for all repeated statements was day or period, with animal(treatment) as a subject for Exp. 1, whereas in Exp. 2, pen(treatment) was used as a subject for DMI and G:F only, and animal(pen × treatment) as a subject for all other analyses following the rationale described by P > 0.68) for molar proportions of acetate, propionate, isobutyrate, butyrate, isovalerate, and valerate, and did not differ between treatments , indicating that animals had similar dietary management before the beginning of the present study.The average daily intake of feed additive was 13.3 ± 0.08, 20.4 ± 0.14, and 20.6 ± 0.15 mg/kg of DM per day for NAR, LAS, and VRM, respectively , which fP ≥ 0.32) for intake and apparent digestibility of nutrients among treatments (P < 0.01) for these variables (P > 0.31) intake and apparent digestibility of nutrients.No treatment × period interactions were detected  for total ruminal SCFA (P < 0.01) total ruminal SCFA values on day 28, 84, and 112 of the study when compared with LAS, VRM, and CON steers, whereas total ruminal SCFA did not differ (P > 0.51) between LAS, VRM, and CON steers (P < 0.01) molar proportion of acetate on day 28, 56, and 112 compared with CON, LAS, and VRM steers, whereas it did not differ (P > 0.25) between LAS, VRM, and CON steers from day 28 to 84. On day 112, steers fed LAS had a lower (P < 0.02) molar proportion of acetate compared with VRM and CON steers, whereas no differences were noted between CON and VRM steers (P > 0.33). Contrary, steers receiving NAR had a greater (P < 0.04) molar proportion of ruminal propionate during the entire experimental period, except on day 84 (P > 0.21), compared with CON, LAS, and VRM steers, whereas LAS steers had greater (P < 0.04) ruminal propionate compared with CON and VRM steers on day 28 and 112, and it did not differ (P > 0.22) between CON and VRM throughout the experimental period. Consequently, NAR steers had the lowest (P < 0.04) Ac:Pr ratio during the entire experimental period, except day 84 (P > 0.16), whereas LAS steers had a lower (P < 0.04) ratio compared with CON and VRM steers on day 28 and 112, and it did not differ (P > 0.34) between CON and VRM throughout the experimental period. No treatment effects were detected (P ≥ 0.27) on the molar proportion of ruminal butyrate, isobutyrate, valerate and isovalerate  rumen pH compared with CON, LAS, and NAR steers, whereas it did not differ (P > 0.84) between CON, LAS, and NAR (P ≥ 0.11) were detected for rumen ammonia (P < 0.01) for these variables.Steers receiving VRM had greater  ADG compared with CON and VRM bulls, and similar (P = 0.17) ADG between NAR and LAS bulls, whereas ADG did not differ (P > 0.28) between LAS, VRM, and CON bulls (P = 0.03) for DMI, being greater in NAR bulls compared with CON, LAS, and VRM bulls, and similar (P > 0.48) between CON, LAS, and VRM bulls. A tendency was detected  for G:F, which was greater (P < 0.02) in NAR bulls compared with CON and VRM, and similar (P = 0.36) between NAR and LAS bulls. However, G:F was also greater (P = 0.05) in bulls receiving LAS vs. CON, it did not differ (P = 0.24) between LAS and VRM, and remained similar (P = 0.49) between CON and VRM bulls. Beginning on day 84, a tendency was detected (P = 0.09) for BW, which was greater (P < 0.02) in bulls receiving NAR compared with CON and VRM, similar (P > 0.13) between NAR and LAS bulls, and it did not differ (P > 0.43) between CON, LAS, and VRM bulls. From day 112 to 140, bulls receiving NAR had greater (P < 0.03) BW compared with CON, LAS, and VRM bulls, whereas BW on these days did not differ (P > 0.51) between CON, LAS, and VRM bulls.Nellore bulls fed NAR had greater  of VRM in forage-based diets, respectively. These outcomes partially resulted from the differences in DMI and ruminal fermentation profile in animals fed NAR. In fact, NAR supplementation in forage-based diets increased DMI by 8.1%, 10.2%, and 11.3% compared with bulls supplemented with CON, LAS, and VRM, respectively. These increments in ADG and DMI for NAR benefited bulls G:F by 18.5% and 12.5% when compared with bulls fed CON and VRM, respectively. Nevertheless, NAR supplementation was the only feed additive that improved final BW by 13.9, 10.8, and 12.1 kg compared with CON, LAS, and VRM bulls fed forage-based diets, respectively. It is well known that ionophores and nonionophores influence ADG, DMI, and G:F of animals fed a high-concentrate diet . When feThe increments in performance parameters of Nellore bulls fed NAR might also be explained by the differences in ruminal SCFA profile, given that altering ruminal fermentation toward propionate and decreasing acetate concentration are positively correlated with greater feed energy utilization and performance . SpecifiVRM is a nonionophore molecule active against gram-positive bacteria by impairing its protein synthesis, resulting in the inhibition of multiplication and, eventually, cellular death . The priIt is well-established that rumen ammonia concentration below 5 mg/dL may limit microbial growth and ruminal fermentation parameters . MoreoveThe impacts of feed additives on apparent nutrient digestibility are still uncertain in the literature . Adding Generally, ruminal fiber digestion is depressed when the ruminal pH declines below 6.2 . In the Improving ruminal fermentation parameters toward propionate production might increase glucose concentration through hepatic gluconeogenic flux . AlthougIn summary, the inclusion of ionophores and nonionophores in forage-based diets did not affect nutrient intake and apparent digestibility of nutrients (Exp. 1). Nonetheless, ruminal fermentation profile (Exp. 1) and intake (Exp. 2) were impacted by the addition of NAR into forage-based diets of Nellore cattle. These results might have contributed partially to the enhanced performance of Nellore bulls receiving NAR compared with CON and VRM cohorts when a forage-based diet was offered. The lack of effects on ruminal fermentation characteristics, ADG, and final BW for LAS and VRM could be attributed to the dose used in the current experiment, despite the manufacture’s recommendation. Nevertheless, this research provides insights into NAR as an important feed additive for forage-based beef cattle diets. Further research should be undertaken to investigate the appropriate dosage of ionophores and nonionophores when used in forage-based beef cattle systems.


The severe acute respiratory syndrome, coronavirus 2 (SARS-CoV-2), is a positive-sense single-stranded ribonucleic acid (RNA) virus contagious in humans and responsible for the ongoing coronavirus disease 2019 (COVID-19) .SARS-CoV-2 mainly spreads via close contact and aerosols or respiratory droplets produced when speaking, breathing, exhaling, coughing, or sneezing . The virIn 67–90% of the patients affected by severe COVID-19, lymphocytopenia occurs, a well-known marker of impaired cellular immunity; both killer T cells and helper T cells have been found to decrease in these circumstances [With the loss of immunosurveillance, latent pathogens in the body can be reactivated, as is the example of herpes viruses. They are a family of deoxyribonucleic acid (DNA) viruses, of which nine are known to primarily infect humans, and five cause extremely common diseases, such as orolabial and genital herpes due to human herpes virus 1 (HHV1) and human herpes virus 2 (HHV2), chickenpox and shingles from human herpes virus 3 (HHV3), and mononucleosis and mononucleosis-like syndrome from human herpes virus 4 (HHV4) and human herpes virus 5 (HHV5) . Over 90Much of the literature discloses infective herpetic reactivations in the course of COVID-19 ,21,22; sIn very rare circumstances of immunodeficiency, e.g., the acquired immune deficiency syndrome (AIDS), they may act synergistically as in the case of EBV-positive HHV8-associated large B cell lymphoma with plasmablastic differentiation , recentlSimilarly, we had previously diagnosed on autoptic specimens an EBV-positive DLBCL involving the whole organism, even the lungs, in a 78-year-old male lymphopenic patient with SARS-CoV-2 infection . MoreoveThe hypothesis that other DNA oncoviruses, such as human papillomavirus (HPV), may also take advantage of the immune system exhaustion induced by COVID-19 is under investigation , as know


This study focuses on Metal Additive Manufacturing (AM), an emerging method known for its ability to create lightweight components and intricate designs. However, Laser Powder Bed Fusion (LPBF), a prominent AM technique, faces a major challenge due to the development of high residual stress, resulting in flawed parts and printing failures. The study’s goal was to assess the thermal behaviour of different support structures and optimised designs to reduce the support volume and residual stress while ensuring high-quality prints. To explore this, L-shaped specimens were printed using block-type support structures through an LPBF machine. This process was subsequently validated through numerical simulations, which were in alignment with experimental observations. In addition to block-type support structures, line, contour, and cone supports were examined numerically to identify the optimal solutions that minimise the support volume and residual stress while maintaining high-quality prints. The optimisation approach was based on the Design of Experiments (DOE) methodology and multi-objective optimisation. The findings revealed that block supports exhibited excellent thermal behaviour. High-density supports outperformed low-density alternatives in temperature distribution, while cone-type supports were more susceptible to warping. These insights provide valuable guidance for improving the metal AM and LPBF processes, enabling their broader use in industries like aerospace, medical, defence, and automotive. Over the past years, Additive Manufacturing (AM), also known as 3D printing, has revolutionised the manufacturing industry, from developing concepts to producing complex, thin, lightweight, and fully functional parts . The joiLaser Powder Bed Fusion (LPBF) is one of the most promising AM technologies and can be directly used to fabricate metal components with high precision and performance for various industries such as aerospace, biomedical, defence, and automotive ,4. In LPHowever, the serious weaknesses of this technology include the construction of overhang structures and the residual stress inherent in the melting and solidification process . In LPBFIn the realm of mechanics and thermodynamics, thermal stress pertains to the mechanical stress generated by alterations in a material’s temperature. To delve into the behaviour of materials during LPBF, a potent tool is Thermal Mechanical Analysis (TMA). TMA is a technique designed to gauge a material’s dimensional changes under carefully controlled heating and cooling conditions, thereby offering valuable insights into both the thermal and mechanical properties of the material . When thSeveral recent studies have used TMA to investigate the thermal expansion and deformation behaviour of materials during LPBF. Chen et al.  developeBased on the literature referenced above, much work has been conducted to predict and evaluate the thermo-mechanical behaviour of metal parts using FEA or similar advanced models and techniques. Either way, it is clear that, to establish LPBF in production technology, extensive knowledge about the transient physical effects during the manufacturing process is mandatory . HoweverIn this research, for the first time, four support types were investigated, conducting 3D printing and computer-aided thermo-mechanical simulations, for the proposal of optimised support geometries that better transmit the heat while maintaining a high quality of the printed part. The optimisation approach focused on the evaluation of the various geometric support parameters for block, line, contour, and cone support structures using the design of experiment methodologies and relevant optimisation algorithms. Based on the findings, along with previously published research conducted by the author , optimisIn previously published work , the autThus, to investigate the behaviour of supported overhang surfaces, small ledge specimens were 3D printed using Selective Laser Melting (SLM) technology, and block-type support structures with configurable geometric parameters were evaluated. For the needs of the experiments, an EOS M290 machine with a Yb-fibre laser of 400 W and a focus diameter of 100 μm was used for fabricating the specimens. The material used was the EOS Titanium Ti64 Grade 5 in powder form: a strong and lightweight Ti6Al4V alloy with a generic particle size distribution of 20–80 μm, and a powder chemical composition of Ti , AI (5.50–6.75 wt%), V (3.50–4.50 wt%), and 1.05 wt% of other elements.The aim of these experiments was to define the minimum and the maximum support parameters that best produce the specimens without significant printing defects and build failures. It was found that low dense support structures were more prone to distort and crack due to high thermal stresses applied in the area, as shown in Moreover, due to the high temperature and thermal stresses applied while printing, it was observed that both high- and low-density support structures had significant effects on the quality of the printed specimen. Specifically, as highlighted in Given the findings mentioned earlier, it became crucial to undertake numerical investigations into the thermo-mechanical responses of the typical support structures utilised in metal AM and LPBF. The objective was to suggest improved support configurations that enhance print quality, even in the face of the extreme thermal conditions during printing. To achieve this, a set of numerical simulations was carried out to cross-reference the results with the experimental data. Initially, the study focused on analysing block support structures, and subsequently, line, contour, and cone structures were investigated in the later stages of the research.To validate the experimental results, a thermo-mechanical simulation was created using COMSOL Multiphysics and a small L-shaped specimen was designed. As observed in the literature, such small ledge overhang geometries are most commonly used in LPBF and metal AM to test and evaluate the performance of the support structures. The specimen’s geometry along with its dimensions and the supports’ design domains are illustrated in Before initiating the thermo-mechanical simulations on the component with the block-type support, preliminary experiments were carried out to establish the primary framework and identify the specific areas for investigation in terms of the thermal and mechanical analysis. The goal was to simulate the LPBF process as much as feasible and record the thermo-mechanical behaviour of the imported assembly. Titanium alloy was used as a material and a heat source of 1550 °C, which is approximately the melting point of titanium, was set between the supports and the part’s overhang surface.All the simulations were performed in COMSOL Multiphysics v6.1, while the 3D models were designed in SolidWorks, San Diego, CA, USA. The integration of specimens and supports from SolidWorks to COMSOL is a straightforward process. This seamless transfer allows for a smooth transition between the two software platforms, enabling users to effectively utilise the features and capabilities of both programs. By importing parts and support from SolidWorks to COMSOL, users can leverage the advanced modelling and simulation capabilities of COMSOL while benefiting from the design and engineering functionalities of SolidWorks. This integration enhances the overall efficiency and effectiveness of the design and analysis process, enabling users to seamlessly work with complex models and simulations.Various materials can be used in metal AM and LPBF systems such as steels (Fe + C +…), aluminium-based alloys (Al + Si + Mg), titanium-based alloys (Ti + Al +…), and nickel-based alloys (Ni + Cr +…). The choice depends on the application, the material properties, and the compatible 3D printing machine. In this study, a grade 4 titanium was selected since its properties are very close to the majority of the titanium alloys used in LBPF and metal AM in general. It is a strong and lightweight Ti alloy with excellent corrosion resistance, a max melting temperature of 1660 °C, and a specific heat capacity of 0.53 (J/g) °C. In the modelling, the modulus of elasticity was assumed to be 105 GPa with a Poisson’s ratio of 0.37 and the thermal conductivity was assumed to be 17.2 W/mK.Regarding the heat source, a value of 1550 °C was set, which represents the melting point of titanium while printing in SLM . The amTo demonstrate the thermal stress, heat distribution, temperature spectrum, and heat transmission from one domain to another, various branches of physics, such as heat transfer in solids and solid mechanics, were employed. These disciplines were utilised to analyse thermal behaviour and illustrate how heat was transferred within a system, including support structures. The analysis revealed both the thermal stress and heat distribution on the part under consideration. To maintain simplicity and focus on the main assembly, the metal powder surrounding the part was excluded from the simulations.inel) linked to that specific layer using the formula: ref” corresponds to the temperature at which zero thermal stress occurs. The coefficient of thermal expansion was considered to be 8.6 × 10−6 (°C)−1 and the temperature applied “T” was established as 1550 °C [In thermo-mechanical modelling, a significant aspect involves examining thermal expansion, which refers to the material’s tendency to alter its volume in response to temperature variations. Within this modelling framework, heat transfer is calculated alongside structural mechanics, which is treated as a combined problem. Throughout the modelling process, it was assumed that the surface of the component in contact with the support maintained a temperature of 1550 °C. Consequently, it became feasible to compute the thermal expansion (ε 1550 °C .ref). Consequently, the heat loss at this location arose from both heat transmission from the support and radiation to the surrounding environment. The modelling process encompassed two boundary conditions: one pertained to the build plate, while the other related to the point where the component connected with the smaller part in contact with the build plate. The supports and the part were made to be uniform to emphasise the contrast more effectively in their heat transfer capabilities.Heat dissipation was also considered, and it was presumed that the entire model was surrounded by a temperature of 21 °C  to be investigated along with their respective levels. Based on the literature and the author’s prior publication , three mDuring the Design of Experiments (DOE), the Response Surface Methodology (RSM) based on Central Composite Design (CCD) was used to perform the experiments and define the different configurations. This method was selected since it is especially useful in the analysis, visualisation, and optimisation of responses. Regarding the CCD setup, a face-centred approach was followed with an alpha value equal to 1, while no replicates of factorial, axial, and centre points were used. As a result, 15 unique alternatives  for this investigation were selected . All the DOEs, the data analysis, the visualisation, and the optimisation of the responses were performed in the Design-Exert v13 software.Thus, for the DOE for block-type supports, the CAD assembly used to set up and run the simulations is shown in After the completion of the thermo-mechanical simulations, in order to evaluate the performance of the support structures, four responses were investigated: support volume, thermal stress, plate temperature, and overhang displacement. The volume of the support structures was measured in SolidWorks. Then, they were exported in STEP format and imported one by one in COMSOL where the simulations were conducted.The thermal stress was measured by recording the maximum value  of the overall stresses applied on the build plate since it was found that the highest values of thermal stress occurred around the build plate where the supports were anchored a. Thus, The temperature distribution was measured by recording the maximum value of the build plate temperature calculated in COMSOL since the overall approach was to investigate the heat transmission and the heat capacity of the various support structures. As shown in The displacement magnitude was measured in terms of the maximum deformation value of the assembly based again on the COMSOL simulations. It was found that the maximum deformation occurred on the specimen’s overhang surface with a positive direction on the z-axis, especially on the edge in front, as illustrated in For the in-depth investigation of block-type supports and their parameters, quadratic models were used to analyse the experiments. According to the ANOVA results, all the factors satisfied the criteria for a well-designed model. Specifically, “F” and “P” values imply that a model and the model terms, respectively, are significant, predicted R² and adjusted R²  indicate a reasonable model agreement, while an Adeq precision  greater than 4 is desirable. Therefore, all the quadratic models were approved for further analysis .The ANOVA showed that tooth height (A), tooth top length (B), and X, Y hatching (C), along with their interactions, had a significant effect on the support volume, thermal stress, plate temperature, and overhang displacement. The correlation between tooth height, tooth top length, and X, Y hatching on the support volume is illustrated clearly in The effect of tooth height, tooth top length, and X, Y hatching on the thermal stress applied on the build plate is shown in The effect of tooth height, tooth top length, and X, Y hatching on the specimen’s overhang displacement is illustrated in Comparing the numerical simulation findings with the experimental work discussed at the beginning of this article, significant similarities were observed. The numerical simulations showed that the highest thermal stress was applied on the build plate as illustrated in As the numerical results for the block-type support were validated against the experimental data, additional numerical simulations were conducted using the same methodology for line, contour, and cone supports, as they are commonly employed in laser powder bed fusion. Line and contour supports share a resemblance with block supports as they both comprise (i) the supporting body, which determines the support’s shape and density, and (ii) the tooth area, which relies on the points of contact between the supports and the overhanging surface of the part. In contrast, cone-type supports consist of individual pillars with adjustable lower and upper diameters. The morphology of block, line, contour, and cone supports is shown in To perform the DOE for line, contour, and cone supports, similar to block-type supports, three main geometric parameters were selected for investigation: line-type—tooth height, tooth top length, and cross line interval a,b; contIt is important to note that all chosen support types can be created using commonly available slicer software, such as Materialise Magics. However, for the purposes of this study, the supports were designed from the ground up to ensure a parametric design approach, thus minimising the risk of structural problems during simulations. Additionally, beyond the specific parameters investigated in this study, each support type possesses a multitude of other parameters that significantly influence their geometry and ease of removal. These parameters were kept consistent, with their values drawn from the existing literature and relevant experimental research ,40. For Similar to the block-type supports, the identical simulation configuration was applied to conduct the thermo-mechanical analysis for line, contour, and cone support structures. The L-shaped specimen, support domain 2, and the build plate area remained unchanged. However, in domain 1, the different support options used during the Design of Experiments (DOE) were individually subjected to thermo-mechanical analysis. An example of the designed line, contour, and cone support structures along with some of their input parameters are illustrated in For the line-type support structures, the effect of tooth height, tooth top length, and cross line interval on the support volume is illustrated in The correlation between the tooth height, tooth top length, and cross line interval on the thermal stress and temperature applied on the build plate are shown in The ANOVA results regarding the overhand displacement and its correlation with the tooth height, tooth top length, and cross line interval are shown in Regarding the contour-type support structures, the findings were also very close to those of the block-type and line-type supports. It was observed that tooth height and tooth top length did not significantly affect the support volume, as illustrated in The effect of the tooth height, tooth top length, and contour offset on the thermal stress applied on the build plate and the plate temperature are shown in The effect of the tooth height, tooth top length, and contour offset on the overhang displacement is illustrated in Regarding the cone-type support structures, the findings displayed a degree of variation when compared to the block, line, and contour supports. This divergence arises from the distinct structure of cone-type supports, which consist of separate pillars with adjustable lower and upper diameters. Consequently, there was no tooth area defined by configurable tooth parameters in cone-type supports. In The correlation between the contact platform diameter, contact part diameter, cone spacing, and the thermal stress applied on the build plate is shown in On the other hand, the plate temperature was greatly affected by the contact part diameter and cone spacing, while the contact platform diameter barely affected the plate temperature. This is illustrated clearly in Regarding the specimen’s overhand displacement, it was greatly affected by the contact platform diameter, contact part diameter, and cone spacing. Based on the plots illustrated in Following the analysis of the ANOVA results and the generation of correlation plots between the input variables and the output measures, numerical optimisation was performed in the Design Expert (v13) software for all four support types. The goal was to determine the optimal parameters for each support type, aiming to minimise the support volume, thermal stress, and overhang displacement, while maximising the plate temperature. The minimisation of the support volume was prioritised to reduce material consumption, resulting in decreased printing time and lower overall costs. Minimising the thermal stress on the build plate was crucial to prevent warping defects and ensure that the supports remained securely attached to the build plate. Similarly, minimising the overhang displacement was essential to maintain a high print quality and prevent defects like warping and curling. Conversely, the plate temperature was maximised since denser supports facilitate improved heat transfer and offer better control over residual stresses in the printed part ,37.To perform the numerical optimisation, a desirability approach was used. Desirability (D) evaluates how closely all the responses meet the assigned criteria and can range from 0 to 1. A “0” desirability score indicates that one or more responses fall outside the acceptable limits, while a “1” desirability score indicates that all the goals are perfectly satisfied . The fou2) was found in cone supports, the minimum thermal stress (3.83 × 109 N/m2) and the maximum plate temperature (872.7 °C) in block supports, while the minimum overhang displacement (0.393 mm) was observed in line supports.The optimal solutions, one for each support type according to the highest desirability, are shown in Based on the numerical results, parameters related to the contact area between the part and supports, and those that define the geometry and density of the supports, play a major role in producing non-defective parts while simultaneously minimising both the printing time and overall costs. It was observed that block support structures effectively meet the optimisation criteria for reducing residual stresses and preventing part distortion, despite their relatively high volume. Subsequently, the findings were discussed and compared with previously published research. Further analyses were carried out on the four responses , and noteworthy observations made during the numerical simulations are presented.The plots of the four support types that illustrate the correlation between the thermal stresses applied on the build plate and the support alternatives are shown in Based on the simulations, apart from the stresses that developed in the vicinity of the supports, a noteworthy amount of stress was observed on the top of the supports, particularly at the points where they contacted the printed part. The maximum stresses were observed on supports with high values of tooth height and low values of tooth top length a and on In The connection between heat transfer and thermal stress is evident, as both factors influence the part’s performance during printing in LPBF at elevated temperatures. In this research, the temperature of the build plate was investigated to assess the effectiveness of supports in terms of optimal heat conduction. According to the simulations in COMSOL, it was found that higher build plate temperatures resulted in supports that better facilitated heat transmission. According to the optimisation findings, this is particularly achievable in high-density support structures. However, it is important to note that such high-density supports also experience higher levels of thermal stress. An example of the supports’ temperature distribution is shown clearly in The displacement plots of the part’s overhang surface for each alternative are shown in Another significant observation that occurred while conducting the thermo-mechanical simulations concerns the deformation of the supports under the high temperatures and thermal stresses developed around this area. As shown in In addition to the geometric parameters of the supports examined in this study, there are other factors that significantly influence the distortion of an overhang surface during the printing process in laser-based technologies. These factors are associated with the heat source, such as laser speed and laser power . These aSignificant similarities were observed when comparing the findings of this research with relevant published work. The optimisation results showed that the optimum support density to maximise the thermo-mechanical performance and reduce the volume is characterised by average spacing values of 1.63 mm for the X, Y hatching for block supports, 1.24 mm for the cross line interval for line supports, 1.4 mm for the contour offset for contour supports, and 1.3 mm for the cone spacing for cone supports. Similar results were observed in previously published research   where, fSimilarities were also observed in Cheng et al.  where vaFor block supports, the optimal solution was characterised by the lowest value of tooth height (1 mm), average values of tooth top length (0.295 mm), and average to high values of X, Y hatching (1.625 mm);For line supports, the optimal solution was characterised by the lowest value of tooth height (1 mm), average values of tooth top length (0.347 mm), and average values of cross line interval (1.242 mm);For contour support structures, the optimal solution was characterised by the lowest value of tooth height (1 mm), average values of tooth top length (0.365 mm), and average values of contour offset (1.402 mm);For cone supports, the optimal solution was characterised by average to high values of contact platform diameter (1.7 mm), the highest value of contact part diameter, and average values of cone spacing (1.27 mm);The average support volume of block-type supports was much higher (approx. 45–50% up) compared to line, contour, and cone support structures, which were based on the same input parameters;Higher thermal stresses were observed on high-density support structures. Also, supports with high values of tooth height and thin features such as thin cone supports were more exposed to high thermal stresses;High-density supports exhibited better temperature distribution compared to low-density support geometries;Overhangs supported by cone-type support structures were more prone to warp. Moreover, supports with high values of tooth height and low lower/upper diameter values were more prone to distort;3) was found in cone supports, the minimum thermal stress (3.83 × 109 N/m2) and the maximum plate temperature (872.7 °C) in block supports, while the minimum overhang displacement (0.393 mm) in line support structures;The minimum support volume  used in metal AM and LPBF. It involved 3D printing, the design of an experimental methodology, and thermo-mechanical simulations to examine the thermal behaviour of 60 distinct support configurations during laser-based 3D printing. The goal was to identify optimal solutions for each support type, ensuring cost-efficiency, minimal material usage, and the production of well-printed parts. The key objectives were to optimise the relief of residual stresses during printing, minimise part and support deformation, and reduce support volume. Based on the analysis and optimisation results, the most important conclusions are as follows:Future work will include further research and experimentation to evaluate the findings regarding the proposed optimised support structures using 3D printing in LPBF. Similar test specimens and practical applications will be employed to assess the thermo-mechanical performance of the supports and the overall print quality. Furthermore, future work will emphasise an investigation into support removability. Developing supports that are easy to remove, minimise material consumption, and maintain high print quality is crucial in the context of metal additive manufacturing, contributing to the potential adoption of LPBF as a production method.


Voluntary male medical circumcision (VMMC) reduces HIV acquisition by up to 60%. Kenya has successfully scaled up VMMC to an estimated 91% of eligible men and boys in certain regions in combination due to VMMC and cultural circumcisions. VMMC as a program is implemented regionally in traditionally non-circumcising counties where the prevalence is still below 91%, ranging from 56.4% to 66.7%. Given that funding toward VMMC is expected to decline in the coming years, it is important to identify what models of service delivery are most appropriate and efficient to sustainably meet the VMMC needs of new cohorts’ eligible men. To this end, we compared the costs of facility-based VMMC and one within a rapid results initiative (RRI), a public health service scheduled during school holidays to perform many procedures over a short period. We employed activity-based micro-costing to estimate the costs, from the implementer perspective, of facility-based VMMC and RRI-based VMMC conducted between October 2017 and September 2018 at 41 sites in Kisumu County, Kenya supported by the Family AIDS care & Education Services (FACES). We conducted site visits and reviewed financial ledger and programmatic data to identify and quantify resources consumed and the number of VMMC procedures performed during routine care and RRIs. Ledger data were used to estimate fixed costs, recurring costs, and cost per circumcision (CPC) in United States dollar (USD). A sensitivity analysis was done to estimate CPC where we allocated 6 months of the ledger to facility-based and 6 months to RRI. Overall, FACES spent $3,092,891 toward VMMC services and performed 42,139 procedures during the funding year. This included $2,644,910 in stable programmatic costs, $139,786 procedure costs, and $308,195 for RRI-specific activities. Over the year, 49%  of procedures were performed as part of routine care and 51%  were performed during the RRIs. Procedures conducted during facility-based cost $99.35 per circumcision, those conducted during the RRIs cost $48.51 per circumcision, and according to our sensitivity analysis, CPC for facility-based ranges from $99.35 to $287.24 and for RRI costs ranged from $29.81 to $48.51. The cost of VMMC during the RRI was substantially lower than unit costs reported in previous costing studies. We conclude that circumcision campaigns, such as the RRI, offer an efficient and sustainable approach to VMMC. Voluntary medical male circumcision (VMMC) reduces female-to-male HIV transmission by 50–60% –3. PriorIn 2007, the World Health Organization (WHO) and United National Agency HIV/AIDS (UNAIDS) set the goal to achieve 80% of males 15–49 years old circumcised by 2020 in 14 priority countries in southern and eastern Africa .In 2016, modeling data from Malawi suggested that scaling up VMMC services to those younger than 15 years of age is an investment for HIV prevention in the future . In respAs countries switch from donor to nationally supported programs, cost-efficient services will help ensure long-term sustainability of VMMC . InnovatHowever, few studies have estimated the costs of different models of VMMC service delivery. In 2012 a study reported VMMC costs around $66 per procedure in Kenya when using multi-site facility sampling . ShortlyIn response to the scarcity of data on the costs of different models of VMMC implementation, we conducted a study to compare the costs per circumcision (CPC) within routine facility-based care and RRI within Kisumu County, Kenya. This study was conducted in collaboration with the Kenya Ministry of Health, Kenya Medical Research Institute, and Family AIDS Care & Education Services, a Center for Disease Control/President’s Emergency Plan for AIDS Relief (CDC/PEPFAR) implementing partner.Facility-based VMMC are conducted at a stand-alone medical clinic where a patient can present for a circumcision anytime of the year. These locations typically have personnel and supplies onsite year-round. Teams conducting these procedures consist of a surgically trained clinical officer, a surgical assistant, an infection control officer, an HIV testing counselor, and outreach worker. Resources include surgical rooms, recovery rooms, surgical supplies, and autoclaves. Activities include infection testing capabilities, surgery, surgical 1 week follow-up, and infection prevention counseling.In 2009, the Kenya National AIDS and STI’s Control Programme (NASCOP) and the Ministry of Health (MOH) introduced RRIs as part of the campaign to increase the number of VMMC conducted throughout Kenya. RRIs typically take place during school holidays to target school-aged boys. During these school holiday campaigns, multidisciplinary teams are assembled, and programs enlist permanent surgical team staff, per diem surgical team staff, and social mobilization personnel to raise awareness about the RRI campaign . During We employed an activity-based micro-costing approach to estimate costs of VMMC conducted within routine care (facility-based VMMC) and within RRIs between October 2017 and September 2018 across Kisumu County, Kenya. We assessed costs associated with VMMC across 41 sites which are supported by FACES in Kisumu County . EthicalWe conducted in-person site visits to define resources used for facility-based VMMC and RRI campaigns. In each site, we conducted informal interviews with in-charge clinicians and VMMC staff to assess the resources used in the facility and during an RRI to support VMMC. The team appraised the supplies used such as anesthesia, surgical tools, cleaning/autoclave, personnel needed for the site to function, per diem staff, travel costs, and utilities.FACES staff collected data from each clinic every month on the number of medical circumcisions conducted by age group as part of routine program reporting. Reporting include categorization of VMMC into 3 different age groups .We reviewed ledger data from the FACES program, payroll from the Kenya Medical Research Institute (KEMRI), and payroll from the Kenya Ministry of Health (MOH) for the duration of our study period from 2017–2018 to identify money allocated for salaries. The exchange rate for the study period (2017–2018) was used to report on spending values from Kenyan shilling (KSH) to United States Dollar (USD).Using the ledger data, we categorized each line item into standard costing categories: 1) Recurrent Goods, which include items that require recurrent purchases and payments; 2) Capital Goods, which include large purchases for equipment used over many years; 3) Facility costs, which include rent of locations where VMMCs were performed; 4) Personnel, which includes MOH, KEMRI and per diem employees who participate in mobilization, HIV testing, surgical procedures, follow-up, transportation, or sanitation; and 5) Services, which include transportation, printing and reproduction .To accurately represent costs per procedure in our intervention, we further categorized all spending into broad categories with a total of 49 different groups including: insurance, office supplies, rent, utilities, value added tax (VAT), facilities, per-diems, transportation, etc. We collected data on the total number of procedures completed from 2017–2018. Total number of procedures were grouped by age categories and by month of completion.The focus of our analysis was on the unit cost of one facility-based procedure compared to an RRI-specific procedure. To estimate the average CPC conducted during routine care and the average CPC conducted within an RRI, we totaled all spending based on three categories: stable programmatic, RRI-specific, and per procedure costs. To account for spending that did not vary by procedure or if it occurred during an RRI, we calculated the total costs and divided it equally across the months within our study period. Next, we took the total spent on procedures-related expenses and divided them by the total number of VMMCs conducted over the year. We then allocated these expenses based on the number of VMMC conducted during each month of the study period.In order to appropriately divide up the stable programmatic costs among the RRI and facility-based months, we accounted for 25% of the stable programmatic costs to be during RRI months and 75% to be during facility-based months since 3 of the 12 months were during RRI and the other 9 were facility-based and the costs should remain stable throughout. We accounted for 100% of the RRI-specific costs to occur during RRI months. Next, we calculated the procedures that occurred during the RRI months  and non-RRI months. We then used the percentages of procedures to compare the per procedure costs during an RRI to facility-based VMMC.Although we expected high numbers of VMMC to occur during school holidays which are in December, April, August, there was an additional increase in VMMC conducted during other months. To address this overflow, we conducted a sensitivity analysis which assumed that RRIs occurred over 2 months instead of 1 to estimate the potential variation in the CPC for facility-based and RRI in this context. To do this, we totaled all spending that varied by procedure and divided it up for 6 RRI months  and the 6 non-RRI months .From October 2017-September 2018 staff conducted a total of 42,139 VMMCs. Of these, 21,514 (51%) were conducted during routine care (facility-based) and 20,625 (49%) were conducted during RRIs. The total number of VMMCs conducted and age distribution of clients circumcised varied by month. Some of the peaks occurred in December, April, and August coinciding with the school holidays and the months RRIs took place . Peaks iOverall, FACES spent $3,092,891 toward VMMC. This included $2,644,910 in stable programmatic costs, $308,195 in costs that were directly related to RRI activities , and $139,786 in per procedure costs .Spending per month during an RRI varied with most of the spending allocated to personnel which accounted for 42.9% of spending and included per diem staff and consultants needed during the outreach . The secWe then conducted a sensitivity analysis for CPC to account for deviation from the predicted values. The total number of VMMCs in November, December, March, April, June, and July were included. CPC for facility-based ranges from $99.35 to $287.25 and for RRI costs ranged from $29.81 to $48.51.Our results show that the average cost of one VMMC in Kisumu County in Kenya during an RRI was $48.51 compared to $99.35 for a facility-based procedure. We also saw a high number of VMMC within childhood cases 10–14 years old in our RRI campaign. A study documenting the first RRIs conducted in former Nyanza province  in 2009 and 2010 reported a lower CPC than we observed, the demand was much higher at the time of rollout. RRI reported costs for VMMC within FACES fell below the range of recently published estimates , 20. A sInterestingly, 51% of the procedures were conducted during RRI, and children 10–14 years made up 70.5% of the total clients. Most of the procedures occurred during the school holiday months and RRIs; however, May and July also had high rates of VMMC due to half term breaks. Among regions/communities with high HIV prevalence that have reached saturation, focus might shift to boys within the 10–14-year-old cohort. Given that cost was less for males 10–24 years old during an RRI, this may serve as an important modality. Additionally, data has shown that targeting this age group is a good investment for HIV prevention prior to sexual activity . HoweverAlternatively, despite the progress that Kenya has made with regards to VMMC, a gap still exists within Kenya in relation to older men, 15+, who represented 29.5% of total procedures. It is likely an integration model of RRI and facility-based outreach where cohorts of boys are all circumcised at the same time every one to three years as saturation is reached and newborns age into the VMMC eligible age category . AnotherAlthough we observed substantial differences in the unit cost of VMMC, our estimates of the unit cost of both RRI and facility-based VMMC are limited. First, we were only able to track VMMC completion by month. Although the RRIs were focused in December, April, and August, increased VMMC also occurred during December, March, and July which coincided with other school holidays. Second, we were not able to track the exact date that specific resources were used. For example, certain payments were made in preparation for an RRI the month that preceded mobilization  while other costs were paid after completion of RRIs (e.g. staff per diems). We attempted to account for these differences by lagging VMMC by one month, however we could not account for all variability. Third, it is likely every individual did not have an equal chance of being a participant in our RRI intervention; younger males had more free time during an RRI as they were during school holidays compared to those who are older and more likely working.The highest number of VMMC were conducted during school holidays and term breaks. The majority of procedures during an RRI and term breaks were conducted in school aged children, 10–14 year old. Increasing capacity to conduct VMMC during these times of the year is an efficient approach to VMMC within this age group until implementation reaches scale. However, as we are conducting VMMC among emerging populations of adolescent and pre-adolescent males, it is imperative we figure out ways to continue engaging those older than 15 years old during holiday campaigns or through other outreach measures. While lower CPC does not ensure sustainability, scale up of RRI may be an efficient model for HIV prevention through circumcision in Kenya.S1 Table(PDF)Click here for additional data file.S2 Table(PDF)Click here for additional data file.S3 Table(PDF)Click here for additional data file.


Neglected tropical diseases (NTDs) are a group of infectious diseases that are common in the tropical regions of the world that include landmasses surrounding the equator such as North America, South America, Africa, Asia, and Australia. NTDs are caused by different microorganisms including bacteria, viruses, fungi, and parasites. Many NTDs involve specific environmental conditions, vectors, and animal reservoirs that favor the survival of microorganisms with complex life cycles. The vast majority of NTDs are caused by parasites followed by bacterial species, fungi, and viruses. Additionally, vector-borne arthropods like mites causing scabies and other ectoparasites can cause NTDs. A list of NTDs, as currently listed by the World Health Organization (WHO), is provided in During the 73rd World Health Assembly, the WHO proposed a road map for the elimination of NTDs by 2030. This mainly aims to control, prevent, and/or eliminate the WHO-listed NTDs . Given tMohapatra et al. reported a cluster of legionellosis (severe pneumonia) cases, including four deaths, in Argentina. Legionella pneumophila is a bacterium that commonly lives in the environment. In COVID-19 patients who suffered from debilitating post-disease syndromes, this opportunistic microbe was found to cause lung infections, especially in hospital settings, leading to nosocomial infections that further deteriorated the patients’ quality of life.During the COVID-19 pandemic, several cases of NTDs re-emerged in various countries worldwide. Mycobacterium tuberculosis), malaria, dengue, measles, and diseases caused by the human immunodeficiency virus (HIV) among others. Additionally, preventive measures such as mass drug administration against filariasis and other parasitic infections were severely affected during the pandemic. Moreover, it was observed that the co-infection of COVID-19 among tuberculosis patients worsened health outcomes and contributed to an increase in morbidity and mortality .The ongoing COVID-19 pandemic not only contributed to the worsening of people’s health, but also affected health-related services that were in place for the management of existing diseases like tuberculosis .Further, Eastern equine encephalitis virus (EEEV) is a zoonotic virus belonging to the family Togaviridae and causes life-threatening encephalitis. It is a vector-borne/arboviral disease that is generally common in the equine population. There is evidence of accidental infections among humans and other vertebrate hosts. It has a high mortality rate, and more than half of infected persons suffer from the sequelae. The first human infection caused by EEEV was reported in Massachusetts, United States in 1938. Since then, sporadic cases of EEEV have been reported from various states confirming their existence in the environment and a re-emergence that accounted for a mortality rate greater than 40%. Despite being confined to the North American region, the virus may spread to non-endemic regions owing to cross-border animal transport and increased globalization .A re-emergence of the Marburg virus (MARV), the causative agent of viral hemorrhagic fever, was also noticed during the prevailing COVID-19 pandemic. MARV belongs to the same virus family group (Filovirus) as the Ebola virus. MARV is a highly pathogenic risk-group-4 virus that results in high mortality (approximately 90%) among infected persons. MARV infection has recently been reported in Ghana. Its re-emergence may be inevitable and could only be controlled with the discovery of an acceptable vaccine and therapeutic drugs which require further research . This enables better preparedness among authorities, minimizes disease transmissibility, and contributes to better control.The recent outbreak of Ebolavirus (EBV) disease in Uganda appears to be the best example of how improved viral disease tracking capabilities allow health administrators and scientists to predict the spread and other clinical and epidemiological characteristic features . Therefore, public health administrators must use genomic surveillance methodologies to understand the viral evolution that could facilitate better preparedness to tackle future pandemic-like situations. Further, vaccine equity  is an Research Topic that needs immediate attention along with improved vaccine that includes/covers recent variants of the virus and booster immunization doses for persons who are prone to re-infections .Given that most diseases globally prevalent can be endemic to certain geographical regions, it is essential to understand their etiology, pathophysiology, epidemiology, diagnosis, management, control, and prevention. The same applies to SARS-CoV-2, which has been continuously mutating and evolving into several viral variants including the more pathogenic Delta and Omicron variants .In a recent observation from Haryana, North India, hundreds of people reportedly presented to the hospitals with acute febrile illness (AFI). Later, diagnostic work-up among 58 of them revealed that the majority of patients were infected with Dengue virus (77.58%), followed by Chikungunya virus (3.44%), Japanese B Encephalitis virus (3.44%), and some had dual infections (2.23%). This study found none of the AFI patients had West Nile fever, scrub typhus, or leptospirosis. The results of this study emphasize the role of laboratory diagnostic methods in identifying the causative microbes during an outbreak or any similar health emergency . It was also observed that knowledge of MPXV was low (45%) among healthcare workers (HCWs). The vast majority (82%) of the HCWs believed that there is a need to learn more about the virus. Among the people who had suffered from COVID-19, many were afraid of MPXV compared to those who did not suffer from the disease .Despite being endemic to Africa, the Monkeypox virus (MPXV) was recently reported in non-endemic regions including the Americas, Europe, and other countries. Interestingly, the current MPXV outbreak was noted to be transmitted majorly through sexual routes. Additionally, the prevailing outbreak of MPXV has been noticed increasingly among HIV seropositive patients probably owing to their abnormal sexual activities , Mycolicibacterium smegmatis (Mycobacteria), MPXV, and MARV . Drugs that have been used to treat MPXV infection include tecovirimat, brincidofovir, cidofovir, vaccinia immune globulin, and trifluridine . Although not currently approved, there are two candidate vaccines in the pipeline against MPXV infection. This Research Topic provides up-to-date knowledge on the re-emergence of NTDs in the context of the COVID-19 pandemic and discusses how such diseases are transmitted and what mitigation strategies should be put in place to control their spread.Among the various strategies that can be employed to tackle emerging, re-emerging, and other NTDs, vaccination and therapeutic drugs assume increased significance. A recent


Arenaviridae, are capable of infecting mammals and are primarily found in rodent reservoirs worldwide. Mammarenaviruses can be transmitted to humans through contact with infected rodents, and though infection is often asymptomatic, some members of this genus can cause viral haemorrhagic fever which has mortality rates ranging from 1% to 50%. These viruses are typically restricted geographically, based on the geographical range of their host reservoirs. Lymphocytic choriomeningitis virus (LCMV) was previously thought to be the only mammarenavirus found across the globe. However, recent discoveries of two novel human mammarenaviruses, Wenzhou Virus (WENV) and Plateau Pika Virus (PPV), in Asia and Southeast Asia show that mammarenaviruses are more widespread than previously thought. This editorial article aims to raise awareness about these emerging viruses, their genetic and ecological diversities, and clinical significance, and to encourage further study of these emerging viruses.Mammarenaviruses, a genus of the family  Sigmodontiae and Muridae. Old World (OW) mammarenaviruses are predominantly found in Murinae and Muridae subfamily rodent reservoirs in Africa and include human pathogens such as lymphocytic choriomeningitis virus (LCMV), Lassa virus (LASV), and Lujo (LUJV) viruses. LCMV is found worldwide due to its natural infection of the common house mouse (Mus musculus) [Mammarenaviruses are enveloped bi-segmented ambisense viruses , capableusculus) . While tusculus) , human musculus) . ImportaLASV alone can cause up to 300,000 infections per year resulting in 5,000 deaths annually, and one out of every three survivors suffer from often irreversible hearing loss . LASV alRattus and Retiviver rats as well as in S. murinus, which are the Asian house shrews [It was previously thought that LCMV was the only mammarenavirus circulating worldwide. In recent years, however, multiple novel mammarenaviruses have been identified in Asia and Southeast Asia. For example, a recent surveillance study done by Li and colleagues tested 351 rodents for mammarenaviral infections via PCR testing and found 42 positive samples for genetic materials indicative of a mammarenavirus. From those positive samples, a novel mammarenavirus, called Wenzhou Virus (WENV), was isolated from the Wenzhou region of China with sequences of the complete small (S) and large (L) genome segments clustered with other known OW mammarenaviruses on the phylogenetic tree . WENV wae shrews .Figure Rattus norvegicus, Bandicota indica, and Rattus exulans) via RT-PCR, with 27 of 627 (4.3%) positive samples from seven locations in Cambodia and Thailand. Similar seropositivity rates of this virus in symptomatic and asymptomatic humans, as well as its detection in geographically disparate rodent species, suggest that WENV has become endemic in these countries.Interestingly, another study revealed similar strains of the OW WENV in Southeast Asian countries, including Cambodia and Thailand, with seropositivity in 89 of 510 (17.4%) samples of patients who tested negative for influenza and dengue but exhibited flu-like symptoms. In the asymptomatic group, a seropositivity rate of 13% was found in 70 of 529 samples from seemingly healthy individuals [Lagomorpha family, in the Qinghai Tibet Plateau region [More recently, another novel mammarenavirus, called Plateau Pika Virus (PPV), has been isolated from plateau pikas, which are small mammals of the u region . Based ou region . RNA sequ region . PPV wasu region .Recent discoveries of novel mammarenaviruses in wider mammalian species, in combination with seropositivity in humans, suggest that the host range for some mammarenavirus infections is wider than previously thought and may be expanding. The wide genetic variations of pathogenic mammarenaviruses could result in increased probability of spillover events with human exposure to many different mammals that can carry these mammarenaviruses in the wild. This increased prevalence of mammarenaviruses in human populations, spillover potential, and their potential to cause severe disease calls for more attention paid to the epidemiological, ecological, virologic, immunological, and pathogenesis investigations into these emerging viral pathogens. These studies will inform the development of prophylactic and therapeutic treatments to protect humans against these novel viruses with pathogenic and pandemic potentials.


The COVID-19 pandemic highlighted the need for better data to understand how ventilation may impact respiratory virus transmission. We evaluated if there was an association between respiratory viruses detected in school room air samples and nasal swabs from students/ staff in the same room.We performed air and respiratory sampling to detect respiratory viruses at 3 time points between Feb 27 - Mar 28, 2023 in 2 elementary schools. Three classrooms, a multipurpose room, and school nurse’s office in each school had 1 air sample/ room collected using Thermo Scientific AerosolSense™ sampler during 8 school hours. Concomitant respiratory viral testing, using self-collected nasal swabs, was performed in students/ staff enrolled in a respiratory virus surveillance school testing program. Air and respiratory specimens were tested for adenovirus (AdV); coronaviruses (CoV) 229E, HKU1, NL63, OC43; human metapneumovirus (hMPV), influenza A and B (Flu), parainfluenza virus 1-4 (PIV), respiratory syncytial virus (RSV), rhinovirus/ enterovirus (RV/EV), and SARS-CoV-2 using the Hologic® Panther Fusion® Respiratory Assay. Detected respiratory viruses in room air and respiratory samples were compared to determine likelihood of capturing current human respiratory viral infections in room air and additional viruses circulating from unsampled students/ staff in the same room. RV and AdV in human respiratory samples were typed by partial genomic sequencing to understand if viruses were similar.Ten rooms, including 6 classrooms with ≈25 people/ room, were sampled at 3 occasions. At least 1 respiratory virus was detected in 27/ 30 (90%) air samples. 59 samples were obtained from 20 students and 5 staff in these rooms; 24 (41%) were positive for ≥1 virus and 11/24 (46%) rooms with concomitant air and respiratory samples had the same viruses detected (Table). RV typing demonstrates that both participants from Room E, Round 1 and 2 had RV-B27. Both participants from Room F, Round 2 with single AdV infections had AdV B3.Concomitant respiratory virus surveillance in air samples and students/ staff can be performed to better understand the role of ventilation and respiratory virus transmission. Additional studies, including sequencing data and symptom assessment, are needed.Rangaraj Selvarangan, BVSc, PhD, D(ABMM), FIDSA, FAAM, Abbott: Honoraria|Altona Diagnostics: Grant/Research Support|Baebies Inc: Advisor/Consultant|BioMerieux: Advisor/Consultant|BioMerieux: Grant/Research Support|Bio-Rad: Grant/Research Support|Cepheid: Grant/Research Support|GSK: Advisor/Consultant|Hologic: Grant/Research Support|Lab Simply: Advisor/Consultant|Luminex: Grant/Research Support