Enterovirus, like the majority of RNA viruses, evolves to survive the changeable environments by a variety of strategies. Here, we showed that HY12 virus evolved to alter its characteristics and pathogenicity by employing a non-synonymous mutation. Analyses of 5′UTR, VP1 and VP2 gene sequences revealed the existence of HY12 virus in an array of mutants defined as quasispecies. The determination of diversity and complexity showed that the mutation rate and complexity of HY12 virus quasispecies increased, while the proportion of HY12 VP1 and VP2 consensus (master) sequences decreased with increasing passages. Synonymous mutation and non-synonymous mutation analysis displayed a positive selection for HY12 quasispecies evolution. A comparison of HY12 virus in different passages demonstrated that HY12 virus altered its characteristic, phenotype, and pathogenicity via non-synonymous mutation. These findings revealed the evolution pattern for HY12 virus, and the alteration of HY12 virus characteristics and pathogenicity by mutation. Enterovirus within the family of Picornaviridae. According to the latest virus classification by International Committee on Taxonomy of Viruses, the genus of Enterovirus consists of 12 species of enterovirus and 3 species of rhinovirus [Virus uses different strategies to survive the changeable environments. Enterovirus, like the majority of RNA viruses, employs the strategies of mutation to adapt the surroundings and implicate in pathogenesis ,2,3,4. Ainovirus ,6. Out oinovirus ,8,9, whiinovirus ,11,12,13The genome of enterovirus contains a single open reading frame (ORF) flanked by 5′-UTR and 3′-UTR . The ORFQuasispecies theory was developed in the 1970s by Manfred Eigen and Peter Schuster to explain self-organization and adaptability of primitive replicons that link genotypic and phenotypic information in the origin of life ,23. ViruVirus mutation occurs during viral replication. Since RNA-dependent RNA polymerase (RdRP) encoded by RNA virus has low fidelity, mutants are easily generated during virus replication ,27,28, wViral mutants persisting in the population can change the tissue tropism, host range, and virus virulence ,35. It hBovine enterovirus infection manifests several clinical signs as such digestive, respiratory, reproductive, and neurological disorders in addition to subclinical infection. Previous studies revealed the genetic and biological variations of BEV strains isolated from cattle manifesting different clinical signs and demonstrated that some BEV strains caused severe digestive and respiratory disease, while other strains caused only mild infection ,39,40. EThe procedures used for this study, including those involving mice, followed a standard protocol reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Jilin University , following strict compliance with the requirements of the Animal Ethics Procedures and Guidelines of the People’s Republic of China. Pregnant ICR mice were obtained from Changchun Biological Products Institute. The mice were maintained in the Laboratory Animal Facility of Jilin Province. All mice had free access to food and water and were kept in a temperature-controlled room (22 ± 0.5 °C) on reverse 12/12 h light/dark cycle. The new-born pups aged three days were randomly assigned to different treatment groups with each cage litter containing a dam and 5–10 pups.l-glutamine in 5% CO2 at 37 °C. After infected by virus, the cells were maintained with DMEM containing 2% FBS, 2 µg/mL gentamycin, and 2 mM l-glutamine.Africa green monkey kidney cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) , supplemented with 10% fetal bovine serum , 2 µg/mL gentamycin, and 2 mM 50 of HY12 virus for each generation were harvested and stored at −80 °C for future use.HY12 virus is an enterovirus isolated from cattle herd manifesting severe diarrhea and respiratory signs . After t50 was determined following a normal procedure and calculated according to the Reed–Muench method [TCIDh method . Plaque h method . A repreTotal RNAs were extracted from cells infected by representative passages of HY12 virus  following procedures as previously described . cDNA sy®-T vector  before sequencing .Amplification of the 5′UTR (P2), VP1, and VP2 genes from representative passage of HY12  was performed by using PCR kit  following the manufacturer’s instruction. Primers for amplification of the 5′-UTR, VP1, and VP2 genes were listed as follows. HY12-VP1-S: 5′-GCGTTGACGATACCTTAAACTA-3′, HY12-VP1-AS: 5′-CTCTGATATGACTGCCATACCT-3′; HY12-VP2-S: 5′-ATCAGCCGCCCAGAACAAGCA-3′, HY12-VP2-AS: 5′-TGTTGGTTGGAAATCAGGAAGAATG-3′; HY12-5′-UTR-S: 5′-CCTTTGTACGCCTGTTTTCCCCACC-3′, HY12-5′-UTR-AS: 5′-ACACGCCCGGAGGTTAGGATTAGCA-3′. PCR-amplified 5′-UTR, VP1, and VP2 genes were either directly sent out for sequencing or cloned to pEGMAlignment analysis was performed using Clustal W methods . VP1 andThe diversity for HY12 quasispecies was determined following the methods described previously . A totalhttp://www.datamonkey.org (accessed on 24 September 2021)) to analyze the ratio of dN/dS for VP1 and VP2 genes. The ratio of synonymous and non-synonymous mutations is used as a criterion for natural selection, where it is considered as positive selection when dN/dS is > 1.0, and negative selection when dN/dS is <1.0.Evolution for HY12 virus quasispecies was analyzed using the methods previously described . Synonym8 TCID50 of P2 and P40 HY12 virus. Control neonatal mice were injected with an equal amount of DMEM medium. Mice were observed every 4–6 h. Lung, intestine, and brain tissues were collected in 14 dpi after mice were euthanized.Pathogenicity for P2 and P40 HY12 virus was comparably determined using the established neonatal mouse model as described . Three gTissues collected from mice infected with P2 and P40 HY12 virus were processed in the procedure described previously . ParaffiQuantitation of viral genome copies in cells infected by P2 and P40 HY12 virus was performed using quantitative real-time PCR following the manufacturer’s instruction. The primers used for real-time PCR were listed as follows. HY12-U: 5′-CAATAACTACTACCCAGAACGGATG-3′; HY12-D: 5′-GTCAGCGAACCCAACAAGATTA-3′. GAPDH was used as calibration control, the primers were listed as follows. GAPDH-U: 5′-GTCTTCACTACCATGGAGAAGG-3′; GAPDH-D: 5′-TCATGGATGACCTTGGCCAG-3′.50 titer, mRNA, and protein expressions were statistically analyzed by the one-way ANOVA test. The results were expressed as means ± standard deviation of the means (SD) from three independent experiments. Results were considered extremely significant when the p value was less than 0.01.Statistical analyses were performed using SPSS Statistics software. TCID50 of HY12 virus (P2) was used to infect the Vero cells cultured in a 60 mm plate. After incubation with the inoculum, Vero cells showed a typical cytopathic effect as early as 6–8 hpi, became rounded, and detached off the flask in 24–48 hpi (50 titer was determined as 10−9/0.1 mL (data not shown). The plaque assay showed a similar morphology with a middle-sized plaque for HY12 virus, indicating the homogeneity of HY12 virus in early passages  were significantly higher than that of P2 virus, indicating that HY12 virus titer significantly increased when the virus was passaged in Vero cells over 40 generations. Similarly, the size of plaque for P20 virus was relatively small, similar to the size for P2 virus. However, it was surprising to note that the sizes of plaque for the representative passages  virus were relatively homogeneous and increased to 30–40 times larger in relation to that in P2 virus (B). These results demonstrated the alteration of HY12 virus characteristic in the course of its passage in cell culture.To explore whether HY12 quasispecies experience any evolution and evolution pattern, P2 HY12 virus was continuously passaged in Vero cells until P120 HY12 virus was harvested. Each passage of HY12 virus was titrated and used as inoculum to infect Vero cells. Representative passages of HY12 virus  were selected to compare their characteristics as such TCID®-T vector, 20 clones for each representative passage were randomly selected, amplified by single colony PCR  sequences decreased with the increasing passage.http://www.datamonkey.org/dataupload.php (accessed on 24 September 2021)) was applied to conduct dN-dS analysis on VP1 and VP2 genes, respectively. Selection pressure on VP1 and VP2 proteins was evaluated according to the value of dN-dS. As shown in To determine whether any regions in VP1 and VP2 genes bear external pressure, analysis software  of P2 and P40 HY12 virus. As shown in p < 0.05), indicating that the mutation R91H in VP1 protein significantly enhanced the efficiency of virus replication.To compare the difference of replication for P2 and P40 virus, qPCR was used to quantitate the viral transcript mRNA level in Vero cells infected by equal amount  were significantly increased in comparison to those of rescued wild-type HY12 virus, demonstrating that R91H mutation is truly responsible for the alteration of HY12 infectivity and the increase of plaque size (data not shown).Mutation of the VP1 gene was demonstrated to change virus binding ability to cells, affect viral infectivity, virulence, and pathogenicity . CompariVP1 is the most variable structural protein for enteroviruses. In relation to other picornaviruses, the capsid protein VP1 of BEV contains five regions lying at or within the so-called canyon  . These f


Uncomplicated urinary tract infections (uUTI) account for a large proportion of primary care antibiotic (AB) prescriptions. This study assessed uUTI-related healthcare resource use (HRU) and costs in US females with a self-reported uUTI.st line/2nd line]/multiple [any line] AB) for most recent uUTI. Multivariable regression modeling was used to compare strata; 1:1 propensity score matching assessed uUTI burden vs matched population .We surveyed US females aged ≥ 18 years who participated in web-based surveys . Participants had a self-reported uUTI ≤ 60 days prior, and took ≥ 1 oral AB for their uUTI. Those reporting signs of complicated UTI were excluded. HRU was measured via self-reported primary care provider (PCP), specialist, urgent care, emergency room (ER) visits, and hospitalizations. Direct costs were calculated as sum of self-reported and HRU monetized with Medical Expenditure Panel Survey estimates. Indirect costs were calculated via Work Productivity and Impairment metrics monetized with Bureau of Labor Statistics estimates. Participants were stratified by number of oral ABs prescribed (1/2/3+) and therapy appropriateness . Participants in the uUTI cohort reported worse absenteeism (+15.3%), presenteeism (+46.5%), overall work impairment (+52.4%), and impact on daily activities (+50.7%) vs NHWS cohort .In total, 375 participants were eligible for this analysis. PCP visits (68.8%) were the most common HRU. Across participants, there were an average of 1.46 PCP, 0.31 obstetrician/gynecologist, 0.41 urgent care and 0.08 ER visits, and 0.01 hospitalizations for most recent uUTI (Table 1). Total mean uUTI-related direct and indirect costs were &1289 and &515, respectively (Table 1). Adjusted mean total direct costs were significantly higher (Table 2) for participants in the ‘2 AB’ cohort vs the ‘1 AB’ cohort , and for the ‘multiple AB’ vs ‘1 AB, 1Table 1. Overall mean uUTI-related healthcare resource use, direct, and indirect cost dataTable 2. Estimated uUTI-related direct costs stratified by (A) number of AB and (B) appropriateness of AB therapy used to treat last uUTITable 3. Mean Work Productivity and Activity Impairment data for uUTI and NHWS cohortsInadequate treatment response, evident by multiple AB use, was associated with an increase in uUTI-related costs, including productivity loss.Jeffrey Thompson, PhD, Kantar Health  Alen Marijam, MSc, GlaxoSmithKline plc.  Fanny S. Mitrani-Gold, MPH, GlaxoSmithKline plc.  Jonathon Wright, BSc, Kantar Health  Ashish V. Joshi, PhD, GlaxoSmithKline plc.


The foamed beads were molded by steam-chest molding using moderate steam pressures of 0.3 to 0.35 MPa independently of the closed cell content. A characterization of the mechanical properties was performed on the molded parts. The steam molding pressure for sintering expanded polypropylene beads with a long chain branched random co-polypropylene is lower than the one usually needed for standard polypropylene beads by extrusion. The energy saving for the sintering makes the entire manufacturing processes cost efficient and can trigger new applications.We explore the foam extrusion of expanded polypropylene with a long chain branched random co-polypropylene to make its production process simpler and cheaper. The results show that the presence of long chain branches infer high melt strength and, hence, a wide foamability window. We explored the entire window of foaming conditions  by means of an ad-hoc extrusion pilot line design. It is shown that the density of the beads can be varied from 20 to 100 kg/m Polymer bead foams are produced by a sintering process  of foamed polymer beads. The sintering process allows us to obtain complex shapes with low density, a unique feature among all the technologies to produce polymer foams . For thiOne of the limitations to ePP application is the low production productivity and the higher price compared to expandable and expanded polystyrene beads (EPS). ePP can be produced with two different technologies; autoclave  or foam extrusion . The extThere has been extensive research on the foaming process of ePP by autoclave  and manyThe foaming of beads by foam extrusion is different from the autoclave foaming. In extrusion foaming, the polypropylene/gas solution passes through small channels (where it is subjected to a high pressure drop rate (PDR), usually much higher than the one experienced in autoclave foaming ) and the2 as blowing agent. The choice of the blowing agent has to comply with not only technical  and economic requirements, but environmental and safety requirements as well. Based on the previous considerations, we selected: (I) isobutane that is a good solvent for PP and its solubility is very high offering the possibility to achieve low density foams [2 has a lower solubility in PP than isobutane, but it is an environmentally friendly choice and it is also more economical than the isobutane. Herein, we present a study on producing expanded beads of random copolymer PP using a commercially available extrusion line. We present insights into the process and the characterization of the foamed PP beads produced by extrusion using isobutane and COty foams ,19; (II)The resulting beads are characterized in terms of density and open cell content. Furthermore, steam chest molding was performed to investigate the molding and mechanical properties of sintered boards.High melt strength polypropylene (HMS PP) was supplied by Borealis and it was used as received. The material exhibits strain hardening at high shear rates comparable with those one in a foaming experiment. The presence of strain hardening ensures a setting mechanism for the growing bubbles that avoids the collapse and the rupture of cell walls. Indeed, it is well known in the literature  that a h2 at a purity of 99.8% were supplied by Linde (Switzerland). The materials used for the experimental campaigns were: (a) RACO PP name WB260HMS from Borealis, Linz, Austria; (b) anti-shrinking agent name Atmer 7300 from Croda, Munich, Germany; (c) nucleating agent name PEA0025793 from Croda, Germany.Moreover, two types of additives were tested: anti-shrinking agent  and a nucleating agent masterbatch of talc microparticles (with a maximum size of 20 microns). The materials were extruded as received. The isobutene and COThe cell structure was analyzed by Scanning Electron Microscope  on the samples cut in nitrogen. ImageJ software was used for shape identification. Density was measured by two methods: (a) multiple beads density: weighting 1 L of beads; and (b) single bead density done by ASTM D792 that is not affected by the beads shape. The open cell content was measured by pycnometer  following the ASTM method D6226 using nitrogen. The average beads size and their sphericity was analyzed by the optical method using a Camsizer . The steam chest molding machine used for sintering the beads was a commercial molding machine Teubert TransTec72/52 PP .Mechanical properties were measured according to ISO 844 for the compression test and DIN 1789 for the tensile tests, using a Zwick Z1485 testing machine  with a displacement of 500 mm/min for the tensile strength test and a Zwick Z050 testing machine  with a displacement of 5 mm/min for the compression test. The test specimens have parallel top and bottom surfaces and essentially perpendicular sides; the dimensions were 50 by 50 by 25 mm.−1, with a fixed strain rate of 1%. The non-linear rheological response of the HMS PP, at high shear rates and deformation, was measured by means of a stress-controlled microcapillary rheometer [The linear rheological response was measured by means of a Physica MCR 301 Rheometer from Anton Paar. The measurements were performed under nitrogen atmosphere using a plate–plate system with a radius of 25 mm (PP25) under oscillatory conditions; the gap between the plates was set to 1 mm. The test sweeps the angular frequency from ω = 0.1 to 50 rad sheometer . The micThe results of density and molding were averaged over at least three samples.www.sulzer.com, accessed on 15 December 2021) optimized for ePP foams. Sulzer’s ePP foaming process consists of a combination of dosing devices, an inter-meshing co-rotating twin screw extruder, a melt conditioning section including melt cooler, melt pump and die plate  temperature sensor equipped with a ceramic insulation close to the measuring tip .η*, as a function of shear rates, is shown in −1. The fitting results have a coefficient of determination equal to 0.8 and show a flow index equal to 0.2, typical of a highly entangled polymer [N1,  [A complete rheological characterization of the polymer is fundamental for designing a stable extrusion foam process of expanded PP beads. The linear and non-linear rheological behavior of HMS PP was investigated at 170 °C. The complex viscosity,  polymer . The obsshown in b, using servoir) . Moreove2 as blowing agents and compared the resulting foamed beads. The PP can be foamed within a broad range of melt temperature and density can be tuned between 100 to 20 g/L. The most relevant pressure for the foaming, the one at the die plate, was kept constant as much as possible between 150 and 200 bar. a and b. As expected, method a (multiple beads density) always gives a lower density than the one measured with method b (single bead density) and the difference is ca. 40%. The insert d in foamT, for five different blowing agent concentrations. For each concentration of blowing agent, we identify an optimum for foaming temperature, foamT is a very important process variable for defining the final foam structure both in terms of density and morphology. A bell-shaped curve typically describes the effect of foamT on the foam density, with a maximum of the expansion ratio at optimal temperature, The branched structure of random copolymer PP molecules exhibits rheological properties, such as high melt strength and rapid crystallization, that assure easy stabilization of the foaming beads. We tested WB260 HMS with isobutane and COay occur ,29,30. AThe effect of an anti-shrinking agent  and a nucleating agent  on the cell morphology of ePP foamed with 7.4 wt% of isobutane was investigated at the optimum foaming temperatures. 2 as the blowing agent (Sulzer 4 and 5). The molding of extruded beads was achieved using steam pressure to 3 bar  and low and medium density beads produced using COThe boards molded as described above were analyzed for their mechanical properties. In The compression test values at 25%, 50% and 75% of compressive strain after the steam chest molding of the board  are shown in No direct comparison between the mechanical properties of boards produced with the described extruded beads and those produced with beads of the same material but produced with the autoclave process, is available. However, compared to commercially available ePP-beads produced with the autoclave method, the described beads reach somewhat lower specific mechanical properties ,32. AutoThe tensile and mechanical compression properties of boards (Sulzer 1–5) are linearly dependent on the final part density of the piece. Furthermore, it was found that there is no significant difference between the mechanical properties of the beads produced with isobutane compared to the ones foamed with carbon dioxide.3. The optimal foaming temperature was investigated by changing the melt temperature and concentrations of isobutene and CO2. The curve of density as a function of temperature has the classical bell shape and the minimum ranges from 140 to 130 °C depending on the gas concentration. In particular, it decreases with the concentration of the blowing agent. The steam chest molding of the foamed beads shows that it is possible to sinter the beads in a wide range of steam pressures (from 0.25 to 0.3 MPa) and a cycle time from 110 to 228 s. The compression test on the molded parts shows that the compression strength at 25%, 50% and 75%  is slightly lower than for the ePP by autoclave reported in the literature. Surprisingly, the sintering of beads can be performed independently on the closed cell content and at only 3 bar steam pressure. The energy saving for the sintering makes the entire manufacturing process cost efficient and can trigger new applications.The foamability of random co-polypropylene by foam extrusion of expanded beads was explored. The results show that all polypropylene grades can be foamed with a fine tuning of processing conditions and the densities can be varied from 20 to 100 kg/m


Zaire ebolavirus. However, effective medical countermeasures, such as vaccines for other filoviruses such as Sudan ebolavirus and the Marburg virus, are presently in early stages of development and, in the absence of a large outbreak, would require regulatory approval via the U.S. Food and Drug Administration (FDA) Animal Rule. The selection of an appropriate animal model and virus challenge isolates for nonclinical studies are critical aspects of the development program. Here, we have focused on the recommendation of challenge isolates for Sudan ebolavirus and Marburg virus. Based on analyses led by the Filovirus Animal and Nonclinical Group (FANG) and considerations for strain selection under the FDA Guidance for the Animal Rule, we propose prototype virus isolates for use in nonclinical challenge studies.The continuing outbreaks of ebola virus disease highlight the ongoing threat posed by filoviruses. Fortunately, licensed vaccines and therapeutics are now available for  Filoviridae family represents a group of filamentous, single-stranded, negative-sense RNA viruses known as the filoviruses [Zaire ebolavirus); Sudan virus or SUDV (species Sudan ebolavirus); Taï Forest virus or TAFV ; Bundibugyo virus or BDBV (species Bundibugyo ebolavirus), Reston virus (species Reston ebolavirus), and Bombali virus  [The oviruses ,2. Only oviruses . Ebolavilavirus) ,2. Of th®, by the FDA, as well as the authorization of Zabdeno® and Mvabea® as a two-dose vaccine by the European Medicines Agency. Importantly, countermeasures were available for testing by the end of the West Africa outbreak and the more recent EVD outbreaks from 2018 to 2020, providing additional clinical data for vaccine efficacy [The largest outbreak of ebolavirus disease (EVD) occurred in 2014–2016, resulting in more than 28,000 cases and 11,000 deaths as a result of infections by the EBOV, which triggered global investments to advance the development of medical countermeasures. For the vaccine field, this ultimately resulted in the licensure of a vesicular stomatitis virus-vectored chimera expressing the EBOV glycoprotein, ERVEBOefficacy ,4. The zThe SUDV has caused seven outbreaks of SUDV disease (SDV) to date, with the most consequential occurring in South Sudan in 2000, infecting over 400 people with a case fatality ratio of 53 percent . PresentFiloviridae family. A case of MVD was identified in West Africa in August of 2021 and the potential outbreak is still being monitored closely as of September 2021. However, through 2020, twelve different outbreaks of MVD have occurred, with a total of 466 documented cases [Marburgviruses are closely related to ebolaviruses in the ed cases , with thed cases . Clinica®, were carried out using the principles of the Animal Rule. Efficacy was demonstrated in both small animal models and in nonhuman primates infected with a lethal dose of EBOV. Because of these efforts, by the end of the outbreak in 2014, a nominal dose that was demonstrated as safe in phase I trials was available for deployment in a ring vaccination strategy [The sporadic and unpredictable nature of filovirus outbreaks requires vaccine development plans based on the FDA Animal Rule pathway. The FDA issued the Animal Rule in May 2002, with the title “New Drugs and Biological Products; Evidence Needed to Demonstrate Effectiveness of New Drugs When Human Efficacy Studies Are Not Ethical or Feasible”. The precepts of the Animal Rule are codified in 21 CFR 314.600 through 314.650 for drugs, and 21 CFR 601.90 through 601.95 for biological products, and updated guidance was provided in 2015 . The aimstrategy .® in the EBOV outbreaks subsequent to 2015 has highlighted the critical role vaccines play as a part of the overall response to filovirus outbreaks. Since approval, more than 300,000 doses of vaccine have been used as part of recent responses to EBOV outbreaks. This has included the vaccination of healthcare workers, front-line responders, and contacts, plus contacts of contacts of index cases. The World Health Organization estimated its efficacy to be 100 percent  [The deployment of ERVEBOThat said, EBOV vaccines were still in preclinical development by early 2014, with the exception of two earlier clinical trials assessing adenovirus serotype 5  and DNA-In the absence of a large outbreak in which clinical efficacy would be demonstrated, the current assumption is that U.S. licensure of these products would occur via the FDA Animal Rule pathway . Under tThe ability of nonclinical testing to provide predictive value for the effectiveness of vaccines relies on several criteria, which are provided in regulatory guidance from the FDA. It is important to appreciate that, under the Animal Rule, animal models are essentially being utilized as a surrogate for humans since clinical trials are not feasible or ethical. These criteria are inherently tied to whether or not the animal species in question is permissive to the challenge agent, and its subsequent pathophysiological and immunological responses are similar to the human responses. The first factor in choosing a challenge isolate is understanding the clinical history of the isolate in terms of the severity of the disease symptoms caused and whether or not the outcome was lethal. In addition to the clinical history of the virus isolate, there are important scientific considerations to ensure the fidelity of the challenge virus over the lifetime of challenge studies, such as limiting the passage history for RNA viruses to minimize genetic drift, the absence of contaminants, and storage under appropriate conditions to preserve potency. Maintaining the viral isolate in a manner avoiding serial passaging is critical to ensuring that the pathophysiological responses in the animal model will be similar to the human disease. The goal is to identify an isolate that (1) mimics the disease course observed in humans with similar measurable endpoints, (2) is relevant to potential isolates that may be observed, and thus (3) will be of use in predicting the efficacy of vaccines in nonclinical challenge models.Although the selection of a viral isolate with the biological properties described above is critical for developing an animal model which can be predictive of clinical benefit, there are technical considerations with regard to the production and storage of the challenge virus that are essential to maintain the applicability of the isolate. The filoviruses are single-stranded RNA viruses, and do not exist in nature as a unique nucleotide sequence, but as a distribution of RNA sequences around a consensus sequence that will change with environmental pressure. Although these viruses primarily circulate in wild mammals in nature, their zoonotic nature arises from the fact that the sequence mixture contains some that are compatible with infecting humans. Therefore, we would expect the initial isolates of an outbreak to be highly virulent, with a genetic drift to lower virulence after coadaptation from multiple passage in humans. The consensus viral sequence for EBOV evolved with extended human passage during the outbreak in 2014 , and speBeyond the typical assays to characterize the challenge stock, it is critical to have full genome sequencing and quantification of the particle:pfu ratio, as both can impact the virulence of the stock . PeriodiEach is a low-passage stock derived from a clinical isolate. This is crucial to avoid genetic drift from selective pressure, which can impact a myriad of product development endpoints.Each isolate is from a known fatal  case.The whole genome RNA sequence is available from both passage 2 and 3.Each isolate has been shown to cause systemic fatal disease in the 1000-pfu intramuscular nonhuman primate challenge model in natural history studies, demonstrating the utility of the model in the evaluation of filovirus countermeasures.Each isolate has a well-documented lineage/pedigree which clearly defines the chain of custody and passage history, including but not limited to cell type used, multiplicity of infection or MOI, media utilized, and technique for harvesting and sequencing.Once the strain of the challenge agent has been selected, a particular isolate of that strain must be chosen for preparation and characterization. Isolate selection is based on several critical criteria:An EBOV virus used in several challenge studies conducted in 2014 to 2016 was isolated from the largest recorded outbreak in western Africa  and was Known isolates of the marburgviruses can be separated into two major lineages (Marburg virus or MARV and Ravn virus or RAVV), but all virus isolates remain members of the same species Marburg marburgvirus. Genomic variation among the marburgviruses is greatest between these two lineages and estimated at 21.0–21.4 percent . Early vH. sapiens-tc/ANG/2005/Angola-200501379 meets the key aspects for a MARV isolate suitable for use as a challenge material in nonclinical testing and evaluation under the Animal Rule. This MARV Angola strain is a human isolate at low passage from a fatal clinical case of an eight-month-old female from Uige, Angola, during the 2005 MARV outbreak [Marburg virus outbreak . The patoutbreak . While tSUDV represents a distinct species among ebolaviruses. SUDV was first identified as the cause of a hemorrhagic disease outbreak in 1976, comprising 284 cases, 53 percent of which were fatal . SimilarH. sapiens-tc/UG/2000/Gulu-200011676 meets the key aspects for a SUDV isolate suitable for use as a challenge material in nonclinical testing and evaluation under the Animal Rule. This SUDV Gulu strain is at low passage from a fatal clinical case. The isolate is from a 35-year-old male from Gulu, Uganda, during the 2000 outbreak. Blood containing the virus isolate was obtained shortly before the patient succumbed to illness. Passage history is documented, with only two passages, and the isolate may be obtained from BEI resources by special request similar to MARV Angola. Similar to MARV Angola, nonclinical vaccine efficacy studies utilizing SUDV Gulu as the challenge stock have been near uniformly lethal in small numbers of mock-vaccinated controls. Natural history studies have also commenced for SUDV Gulu, and to date, have confirmed near-uniform lethality at a 1000-pfu intramuscular challenge. Clinical disease manifestations have been similar to those observed in humans (unpublished data). This study report will also be submitted to the FDA/Center for Biologics Evaluation and Research for cross-reference by sponsors under a Type V Master File.Sudan virus The development of products under the FDA Animal Rule requires well-characterized and understood animal models with well-defined challenge virus isolates. In order to be consistent with the requirements of the FDA Animal Rule, the challenge agent should be a low-passage isolate from a human fatal case that produces an infection in the animal model, that can be used to evaluate medical countermeasures intended to treat the authentic human disease. The isolates for MARV and SUDV are most consistent with these requirements. Given the likelihood that vaccines against MARV and SUDV will proceed towards licensure via the FDA Animal Rule, we recommend at this time that the primary focus, in terms of nonclinical efficacy studies, should be on the Angola and Gulu isolates for MARV and SUDV noted above. The adoption of these challenge isolates will allow the standardization of vaccine development across programs and will simplify the interpretation of nonclinical efficacy studies for both funding and regulatory authorities. Barring any large outbreaks of MARV or SUDV in the near term, the MARV Angola and SUDV Gulu isolates represent recent virus isolates that have well-documented clinical and laboratory histories, meet the guidelines of the FDA Animal Rule, and have demonstrated clinical disease endpoints in nonclinical studies that are consistent with human disease. The Biomedical Advanced Research and Development Authority (BARDA), in coordination with the NIH and DOD, is supporting natural history studies of MARV Angola and SUDV Gulu. Product sponsors will be able to cross-reference Type V Master Files for these natural history studies under their respective regulatory submissions.We have concentrated on describing the identification of the appropriate SUDV and MARV isolates for product evaluation under the FDA Animal Rule in this manuscript because prophylactic and therapeutic countermeasures against these viruses is a BARDA priority. We believe the same considerations used to select the SUDV and MARV isolates will be applicable if additional filovirus strains such as the Bundibugyo or Tai Forest viruses become BARDA priorities for medical countermeasure development or if new and novel Ebola virus strains arise from environmental exposures and become a medical priority.Bombali ebolavirus [The zoonotic nature of filoviruses and the sporadic nature of outbreaks suggests that new and equally lethal versions may emerge. Thus, it may be appropriate to obtain samples from new or previous outbreaks for testing to ensure the effectiveness of medical countermeasures against different isolates. In that light, continued biosurveillance will be a key aspect of ongoing filovirus research, which has recently uncovered a potential sixth species of ebola virus, olavirus . It will


Increasing attention has been paid to the hemodynamic evaluation of cerebral arterial stenosis. We aimed to demonstrate the performance of angiography-based quantitative flow ratio (QFR) to assess hemodynamic alterations caused by luminal stenoses, using invasive fractional pressure ratios (FPRs) as a reference standard.Between March 2013 and December 2019, 29 patients undergoing the pressure gradient measurement of cerebral atherosclerosis were retrospectively enrolled. Wire-based FPR was defined by the arterial pressure distal to the stenotic lesion (Pd) to proximal (Pa) pressure ratios (Pd/Pa). FPR < 0.70 or FPR < 0.75 was assumed as hemodynamically significant stenosis. The new method of computing QFR from a single angiographic view, i.e., the Murray law-based QFR, was applied to the interrogated vessel. An artificial intelligence algorithm was developed to realize the automatic delineation of vascular contour.r = 0.879, P < 0.001; mean difference (bias): −0.006, 95% limits of agreement: −0.198 to 0.209, respectively). Intra-observer and inter-observer reliability in QFR were excellent . For predicting FPR < 0.70, the area under the receiver-operating characteristic curves (AUC) of QFR was 0.946 . The sensitivity and specificity of QFR < 0.70 for identifying FPR < 0.70 was 88.9%  and 85.0% . For predicting FPR < 0.75, QFR showed similar performance with an AUC equal to 0.926.Fractional pressure ratio and QFR were assessed in 38 vessels from 29 patients. Excellent correlation and agreement were observed between QFR and FPR [Computational QFR from a single angiographic view achieved comparable results to the wire-based FPR. The excellent diagnostic performance and repeatability empower QFR with high feasibility in the functional assessment of cerebral arterial stenosis. Large artery atherosclerosis is a leading cause of ischemic stroke, especially in Asian populations, accounting for 30–50% of ischemic strokes in Asia . PivotalCurrently, imaging risk assessment is primarily based on the severity of anatomical stenosis, rather than functional significance. However, there is frequent discordance between anatomical stenosis and functional severity  due to cFractional flow reserve (FFR) is the guideline-recommended standard for assessing the hemodynamic significance of coronary artery stenosis . NeverthQuantitative flow ratio (QFR) was calculated based on combined geometrical data and hemodynamic boundary conditions derived from coronary angiography . QFR hasTherefore, we aimed to demonstrate the feasibility of applying the novel QFR to assess the hemodynamic significance of cerebral artery stenosis, using invasive FPR as a reference standard.This study enrolled patients who underwent pressure gradient measurement of cerebral atherosclerosis stenosis from March 2013 to December 2019. The exclusion criteria were as follows: (a) patients with tandem extracranial and/or intracranial stenosis, (b) extremely tortuous cerebrovascular, (c) extreme foreshortening and overlap of lesion vessel in the angiographic view, and (d) patients with intracranial tumor, arteriovenous malformation or aneurysm. This study was approved by the ethics committee of Jinling Hospital, and each patient was fully informed with written informed consent.The interventional procedure and pressure gradient measurement method were described in our published study . In brie3 and 0.0035 kg/(m⋅s), respectively. Additionally, the QFR computations were performed based on the empiric mean flow velocities   by two independent experienced analysts blinded to FPR data. The image with the lesion segment fully expanded and clearly delineated was selected as the analysis frame. The detailed computational algorithms of the QFR have been described previously . They ca of MCA) . FinallyR2) and root mean square errors (RMSE) were calculated to assess the models. To check the intra-observer and inter-observer reliabilities in the QFR analyses, repeated analyses were performed on all subjects. The agreement of the repeated data was evaluated using the intra-class correlation coefficient (ICC).Correlation analyses were detected by Spearman’s correlation test. Bland–Altman analyses were conducted to compare the agreement of different continuous variables. Generalized additive models were used to investigate potential non-linear relationships . CoefficSince there is no acknowledged cut-off value of FPR in cerebral arterial stenosis, two empiric cut-offs (FPR = 0.70 and FPR = 0.75)  were setP-values of less than 0.05 were considered to be statistically significant.Statistical analysis was performed using MedCalc  and R statistical software . Two-sided A total of 29 patients  were included in this study. There were 25 lesions located in the anterior circulation and 4 in the posterior circulation . Among tAn illustrative case was presented in r = 0.879, P < 0.001) with FPR (P = 0.368). Excellent intra-observer and inter-observer reliability in QFR was confirmed, with an ICC of 0.996 and 0.973, respectively.Quantitative flow ratio showed good correlation , however, which were smaller than that of QFR and FPR. R2 between the percentage of the stenosis (DS% or AS%) and the FPR had changed considerably, indicating a non-linear relationship between them. However, these parameters between QFR and FPR change negligibly, suggesting a linear relationship.The correction between DS% or AS% and FPR were significant  vs. 0.903, 95% CI , nificant . SimilarIn this study, the novel computational QFR derived from a single angiographic view based on artificial intelligence algorithms achieved comparable results to the invasive FPR. Principal findings in this study are summarized as follows. First, excellent correlation and agreement can be seen between QFR and invasive FPR. Second, the computation of QFR shows high repeatability. Most importantly, QFR has excellent diagnostic performance in identifying hemodynamically significant stenosis.The novel computational QFR in our study has comparable results to the invasive FPR. In previous studies, QFR computation was based on the three-dimensional (3D) geometry reconstruction of two angiographic views with an angle difference of at least 25° . NeverthAlthough the results were similar between QFR and FPR, some discrepancies were observed among certain patients. Several possible explanations might account for the deviation. First of all, the length of the vessel segment measured by the pressure wire was greater than that reconstructed in the QFR computation . There mOur study validated the excellent diagnostic performance of QFR in identifying hemodynamically significant stenosis. Although, the diagnostic performance of QFR was better than that of DS% but did not reach statistical significance. Besides the small sample size, only the analysis of localized lesions may also contribute to the results. QFR was considered to have better prognostic performance than conventional angiographic assessment in tandem or multiple lesions . Hence, Moreover, the automatic delineation of vessel contour based on an artificial intelligence algorithm facilitates rapid and stable QFR computation, enabling intraoperative immediate assessment. Although FFR is the gold standard for the functional evaluation of coronary stenosis, the inherent risks of invasive procedures and costs of guidewires limit its widespread use . Wire-frThis study was subject to certain limitations. First, there was only a small validation set of FPR data available to assess the performance of QFR in this pilot study. Second, QFR computation can only be applied to fully exposed lesions rather than the entire vessel due to cerebrovascular tortuosity. In addition, potential changes in downstream vessel resistance induced by cerebral autoregulation also need to be taken into account.Computational QFR derived from a single angiographic view achieved comparable results to the wire-based FPR. The excellent diagnostic performance and repeatability empower QFR with high feasibility in the physiological assessment of cerebral arterial stenosis. Further studies are warranted to demonstrate the specific clinical significance of QFR for patients with cerebral artery stenosis.The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding authors.This study was approved by the Ethics Committee of Jinling Hospital. The patients/participants provided their written informed consent to participate in this study.KH, JD, and XL made contributions to conception and design. KH, WY, RL, YH, and WZ contributed to the acquisition of the data. KH, WY, YC, and ST contributed to analysis and interpretation of the data. KH, WY, RY, RL, and JD contributed to the drafting of the manuscript. JD, KH, ST, YC, RY, and XL were involved in critical revision of the manuscript for important intellectual content. All authors contributed to the article and approved the submitted version.ST received research grants from Pulse Medical Imaging Technology. YC is a full-time employee of Pulse Medical Imaging Technology. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In this work, a comprehensive model for the viral progression in the pharynx has been developed. This one-dimension model considers both Fickian diffusion and convective flow coupled with chemical reactions, such as virus population growth, infected and uninfected cell accumulation as well as virus clearance. The effect of a sterilizing agent such as an alcoholic solution on the viral progression in the pharynx was taken into account and a parametric analysis for the effect of kinetic rate parameters on virus propagation was made. Moreover, different conditions caused by further medical treatment, such as a decrease in virus yield per infected cell, were examined. It is shown that the infection fails to establish by decreasing the virus yield per infected cell. It is believed that this work could be used to further investigate the medical treatment of viral progression in the pharynx. Viruses are inherently related to the existence of many diseases such as hepatitis C (HCV), hepatitis B (HBV), AIDS (HIV), influenza, etc. How a viral infection will progress in the human body depends on many factors, such as the rate of spread of the agent, the immune response, what treatment may be applied, etc. . In ordeThe biological principles regulating dynamic variations in viral load and clinical symptoms can be understood and quantified using mathematical modeling. Parameters that measure the interactions between the virus, its host, course of a disease, and its treatment outcomes can be determined by fitting models to viral load data ,15. OverTo examine the localization and transmission of influenza A virus infections in the human respiratory tract, partial differential equations were used to construct a mathematical model . The priA considerable number of viruses, such as influenza, enters the human body through the respiratory system. The pharynx is the part of the throat behind the mouth and nasal cavity, and above the esophagus and larynx . The phaMany viruses can replicate in the cells that line the respiratory system. Mucus and cilia on the epithelial cells protect the respiratory tract’s surface. Inhaled viruses are retained in mucus, transferred to the pharynx by ciliary motion from the airways and nasal cavity, and then swallowed or coughed up. Droplets frequently travel to the trachea and bronchioles, where they become stuck in the mucus blanket. Droplets can be inhaled directly into the lungs, and some can make it to the alveoli, where virus particles can directly infect alveolar epithelial cells, resulting in viral pneumonia .The aim of this work is to investigate how a virus progresses in the human pharynx by using simple comprehensive mathematical modeling. This work is organized as follows: in the theoretical section the governing equations for the spread of a virus in human pharynx along with the initial and boundary conditions are given, and in the results and discussion section, simulations are presented. Finally, conclusions are drawn and ideas on how this work could be applied to the recent pandemic of COVID-19 are presented.The virus, after entering the first part of the pharynx, will move by both convective flows and diffusion to the lower part of the pharynx and simultaneously increase its population by infecting healthy cells. This problem from an engineering point of view is a diffusion problem coupled with chemical reactions. In particular, it was assumed that only the virus species diffuse in the pharynx. This work closely follows the extracellular model of Rawlings and coworkers ,29. TheiMoreover, convective flow of virus was considered and a virus clearance reaction was also considered, by following Quirouette et al. . In partInfection:Virus propagation:Virus clearance:Uninfected cells accumulation:D stands for virus diffusion coefficient, C represents various species concentration, t is the infection time, v0 is the convective flow velocity, and z stands for axial distance. Subscript infc represents infected cells and subscript unc represents uninfected cells. Subscripts vir and sub represent the virus and the substrate, respectively.Based on the above mechanism, the resulting mass balances are written as follows ,28,29:, the dimensionless position η (η = z/L0), the dimensionless time τ (τ = Dt/L02), the ratio of infected cells to initial cells X1 (Χ1 infc/Cunc0= C), and the ratio of uninflected cells X2 (Χ2unc/Cunc= (C0), the above equations are written as:L0 stands for pharynx length, D0 is a scaling parameter with the diffusion coefficient as a unit, and subscript 0 represents initial conditions. One could directly derive Equations (4)–(6) from Equations (1)–(3) by using the dimensionless quantities previously introduced.The positive sign of the virus convective term shows that the virus physically moves upwards with velocity η = 0) up to the larynx (η = 1). The following initial and boundary conditions were considered:In this work, the pharynx extends from the nasal cavity (The most adverse case of a closed system after infection was considered by applying Equation (7). Alternatively, open boundary conditions  could beThe governing Equations (4)–(6) along with the initial and boundary conditions (7)–(9) were simultaneously solved by using the Galerkin Finite Element Method (GFEM). The system of algebraic equations resulting after weighting with quadratic basis functions and applying the divergence theorem as well as the boundary conditions was numerically solved by Newton’s method. A homemade code written in FORTRAN was utilized for the simulations. A complete description of the method is given in full detail elsewhere ,32,33. AL0) was set equal to 0.15 m, which is a typical value for an adult. The scaling parameter D0 was set equal to 10−7 m2/s. To account for convective flows in the pharynx, the velocity v0 was set equal to 40 μm/s [−12 m2/s, which is in close agreement with the values for this coefficient reported by other workers in the field [u10 was set equal to 7.5 × 10−2 for the whole computational domain [k1uncC0 and k2 were set equal to 1.4 × 10−6 s−1 and 1.6 × 10−5 s−1, respectively. The uncC0 was set equal to 3.8 × 107 cells/cm3 [In this work, the pharynx length  is a constant. This allows us to use a combined kinetic rate constant (k4subC) for uninfected cells’ accumulation reaction.The aim of this work is to study the progression of the virus in the pharynx by using a minimum number of adjustable parameters. Therefore, it was assumed for simplicity that the concentration of the substrate  of eight orders of magnitude is reached within three days after infection. Moreover, a reduction of relative virus load (u1/u10) to six orders of magnitude at six days after infection was considered. Finally, a value of viral relative load (u1/u10) of three orders of magnitude at one day after infection was also considered. The resulting values for the Y, k3, and k4subC are 21.4, 1.25 × 10−4 s−1, and 10−4 s−1, respectively. The value for k3 is in the same order of magnitude with the reported value by Quirouette et al. [−5 s−1). The reported value in this work for Y: 21.4 could be surprising at first glance; this relatively high value could be attributed to the fact that the same population of cells is present during the whole infection time, or in other words, death of cells is caused not only by infection but also by the inherent differences in the parameters of various literature models.The following values for the Y, e et al.  (6.1 × 1The simulations obtained are depicted in w/v) for short washing times [In ng times . Lower cX1 and X2 as a function of infection time. It is shown that X1 and X2 initial exponentially increase with infection time until the point of maximum virus concentration  and X1 is illustrated in The effect of the initial virus concentration on the virus concentration (The main effect of decreasing the yield of virus per infected cell is depicted in k3) by an order of magnitude has very little effect on the maximum infection. This is attributed to the fact that the maximum virus value is mainly controlled by other reactions, such as infection, virus propagation, and uninfected cell accumulation. The value of the virus clearance kinetic constant is the controlling step only in the initially steps of infection, where there is a relatively small number of infected cells (see In ells see  and afteells see .k4subC) is illustrated. It is shown that a decrease in the combined kinetic rate constant for uninfected cells accumulation (k4subC) by an order of magnitude has a strong effect on infection. This could be explained from the fact that lower values for this kinetic rate constant could lead to smaller values of produced uninfected cells, causing a further decrease in the peak virus concentration.In k1uncC0) and virus propagation (k2) is illustrated. In particular, it is shown in both figures that an increase by one order of magnitude of each kinetic rate constant could cause a further decrease on the maximum value of virus concentration. This could be explained by the fact that the infection and the virus propagation reactions are further accelerated, causing the maximum value of the virus concentration to appear at shorter infection times, where the number of uninfected cells is rather small due to the cell accumulation reaction.Finally, in Regarding viruses having a long incubation period (days) before symptoms start to show , this stIn this work, a comprehensive framework for modeling viral progression in the pharynx was developed. The effect of using a sterilizing agent such as an alcoholic solution for washing the pharynx was studied using a numerical experiment. It was shown that the use of a sterilizing agent has no effect on the progress of a virus in the pharynx. Moreover, it is shown that additional treatment with a medicine causing a reduction in virus yield per infected cell has a strong effect on the virus propagation. This study could support that additional medical treatment could be applied in addition to other measures, such as protective equipment , social distancing, lockdowns, etc., in order to reduce the rate of population infection, and thus, lightening the burden on health care systems in developing countries in case of a pandemic such as the COVID-19 pandemic. It is also believed that this work could be used to further investigate the medical treatment of viral progression in the pharynx.


Rousettus aegyptiacus) that when infected, sheds virus in the highest amounts in oral secretions and urine. Being fruit bats, these animals forage nightly for ripened fruit throughout the year, including those types often preferred by humans. During feeding, they continually discard partially eaten fruit on the ground that could then be consumed by other Marburg virus susceptible animals or humans. In this study, using qRT-PCR and virus isolation, we tested fruit discarded by Egyptian rousette bats experimentally infected with a natural bat isolate of Marburg virus. We then separately tested viral persistence on fruit varieties commonly cultivated in sub-Saharan Africa using a recombinant Marburg virus expressing the fluorescent ZsGreen1. Marburg virus RNA was repeatedly detected on fruit in the food bowls of the infected bats and viable MARV was recovered from inoculated fruit for up to 6 h.Marburg virus (MARV), the causative agent of Marburg virus disease, emerges sporadically in sub-Saharan Africa and is often fatal in humas. The natural reservoir for this zoonotic virus is the frugivorous Egyptian rousette bat ( Filoviridae and was discovered in 1967 after an outbreak of Marburg virus disease (MVD) that occurred among German and former Yugoslavian scientists that had worked with MARV-infected non-human primates (NHPs) imported from Uganda in the days prior to becoming ill [Marburg virus (MARV) is the prototype member of the family ming ill . How theming ill ,4,5, witming ill  includinRousettus aegyptiacus) [Marburgvirus (MARV and Ravn virus (RAVV)) RNA and antibodies were first identified in bats in Gabon ,11 and Dptiacus) ,16. Thesptiacus) , Kenya [ptiacus) , and Souptiacus) ,20,21. Mptiacus) . Since the discovery of ERBs as the marburgvirus natural reservoir, research efforts have focused on understanding how MARV is maintained in nature and how the virus spills over into humans. These efforts have included experimental infection studies using captive bred ERBs, first reported by Paweska et al.  and thenWhen considering either route of marburgvirus shedding, saliva, or urine, environmental contamination of surfaces could also represent a means for MARV bat-to-bat or bat-to-human transmission beyond direct biting or deposition of urine in mucous membranes of susceptible hosts. Further, the environmental stability and viability of the pathogens must be considered ,31,32,33To assess the potential of MARV to be transmitted to humans through environmental contamination, specifically contaminated fruit, we tested uneaten and discarded fruit collected from the cages of ERBs experimentally infected with MARV from 5 to 14 days post-inoculation (DPI). This specific time interval coincided with peak viral shedding from the oral mucosa ,26. We fThis fruit inoculation experiment was performed in conjunction with a concurrent study involving MARV-experimentally inoculated ERBs . All exp4 tissue culture infectious dose (TCID50) of MARV  in stern = 3) were swabbed using one set of polyester-tipped applicators, and discarded fruit and fruit spats on each cage floor were swabbed using a second set of polyester-tipped applicators. The polyester-tipped applicators were placed into 15 mL conical tubes containing 1 mL of growth medium , 100 units/mL penicillin, 100 µg/mL streptomycin, 50 μg/mL gentamicin, and 2.5 μg/mL amphotericin B), vortexed, and then centrifuged at 1000× g for 10 min. The supernatants were treated with 50 µL of a 5X antibiotic/fungizone additive and incubated for 1 h at room temperature. After incubation, 100 µL of each antimicrobial-treated supernatant was placed into a well of a deep-well plate containing 500 µL MagMax lysis buffer solution spiked with 0.25 μL of gamma-irradiated Rift Valley fever virus , and the remaining volume of each supernatant was reserved for immediate virus isolation attempts. Statistical analysis was performed, and graphs were produced using GraphPad Prism .Each morning at 7:00 am from 5 to 14 DPI, approximately 13 h from when we estimate the bats began feeding, uneaten bat fruit mix, hereafter referred to as “bat mix” , left in the food bowls of each cage (5 TCID50), medium (1.00 × 103 TCID50), and low (1.00 × 101 TCID50) doses of replication-competent, infectious recombinant (r) MARV expressing the fluorescent ZsGreen1 (ZsG)  contained within 12-well tissue culture plates  were inoculated in triplicate with 250 μL of high , genrocedure , 100 µL 10TCID50 eq/mL were interpolated from a standard curve generated from serial dilutions of the respective virus stocks with known titers in sterile media. It is possible that titers can vary slightly due to the fact that the standard curve was not generated using a saliva matrix. RNA was extracted from the (1) MARV-experimentally infected bat cage fruit samples, (2) oral swabs collected from the MARV-experimentally infected bats, and (3) rMARV-ZsG-inoculated fruit samples using the MagMAX Pathogen RNA/DNA Kit  with the MagMAX Express-96 Deep Well Magnetic Particle Processor  following previously described procedures . Reverse2 flasks containing 2 mL of Maintenance Media with 850 µL of the antimicrobial-treated supernatants, cultures were incubated at 37 °C/5% CO2 through 14 DPI. Cell culture media was replaced with 7.5 mL of fresh Maintenance Media at 1 DPI, and all cultures were tested by immunofluorescent assays at 7 and 14 DPI following published procedures [Virus isolation was attempted on all fruit samples collected from the cages of MARV-experimentally infected bats. Isolations were not attempted on oral swabs due to the large amount of data already published on oral swabs from MARV-infected bats ,25,26,42ocedures .2. Cell culture media were replaced with 2 mL of fresh FluoroBrite Maintenance Media at 1 DPI. At 4, 5, and 14 DPI, each cell culture plate well was viewed under a fluorescence microscope to determine the presence of infectious rMARV-ZsG as indicated by cells containing green foci.Virus isolation was attempted on all rMARV-ZsG-inoculated fruit samples. One hundred and ninety microliters of each antimicrobial-treated supernatant was added to 90% confluent Vero E6 cell monolayers in 12-well tissue cultures plates containing 760 µL of FluoroBrite Maintenance Media  and incubated at 37 °C/5% CO10TCID50 equivalents (eq.) per mL of fluid. MARV RNA positive oral swabs  were collected from all inoculated bats between 5 and 14 DPI  and the other on 11 DPI (0.94 × 100 TCID50 eq/mL). All the samples collected from the cage floors tested negative for MARV RNA. The RVFV internal extraction control failed to be detected in 17.2% (5/29) of the bat food bowl samples and 82.8% (24/29) of the fruit samples collected from the cage floors, possibly indicating the presence of inhibitory substances that may have interfered with RNA extraction or qRT-PCR. After 10 days of daily sampling each morning, two of the 58 (3.45%) samples collected from fruit in the bat food bowls were positive for MARV RNA: one on 6 DPI , medium (1.00 × 103 TCID50), and low (1.00 × 101 TCID50) doses of rMARV-ZsG followed by sampling at 0, 1, 6, and 24 h. In this experiment, a total of 75/108 (69.4%) rMARV-ZsG-inoculated fruit samples were positive for MARV RNA  of banana and mango samples but failed to be detected in 52.8% (19/36) of the bat mix samples , a more rigorous virus viability experiment was performed in which different varieties of fruit were separately inoculated with high  room, immediately followed by bat husbandry activities consisting of cleaning and replenishing food bowls and placing new plastic liners on cage floors. The ABSL-4 room holding the infected bats was on a 12 h light and dark cycle, beginning at 6:00 am and 6:00 pm, respectively. The bats typically eat after the lights go out, indicating that a period of up to 13 h could have lapsed between when the bats ate the fruit and the collection of samples the next morning. Although if bats did some feeding during the day, this time frame could be up to 24 h.4 TCID50 eq/mL but have been as high as 2.5 × 105 TCID50 eq/mL in previous MARV experimental infection studies [1 TCID50 eq/mL [Despite the low number of fruit bowl (bat mix) samples with detectable MARV RNA, oral swab data from this and previous MARV experimental infection studies indicate that bats shed MARV through saliva in concentrations encompassing the range of doses we used for the artificial fruit inoculations. MARV oral shedding loads in this study peaked at 3.21 × 10 studies . Moreove50 eq/mL . This de50 eq/mL ,46. Surprisingly, MARV RNA was detectable for up to 24 h in banana and mango, suggesting that elapsed time may be less of an issue than initially thought. Moreover, rMARV-ZsG isolates were recovered up to 6 HPI in samples of banana and mango, demonstrating the potential for viral persistence on common African fruit.The 6 h persistence of MARV on banana and mango was notably shorter than the 4–5 day persistence of MARV on contaminated surfaces  reported in  and the Historically, MARV spillover to humans has occurred after exposure to areas around bat habitats, such as caves or mines, but not necessarily involving direct contact with bats . To reitIn conclusion, we demonstrated that MARV-infected ERBs can shed virus onto discarded fruit. Further, we demonstrated that infectious MARV is stable for at least 6 h on two types of fruit  routinely consumed by both ERBs and primates, including humans, throughout sub-Saharan Africa. Together, these findings suggest that the consumption or handling of fruit test-bitten or spat-out by ERBs is a risk factor for MARV spillover into to humans and MARV-sensitive wildlife.


Acute and chronic besnoitiosis in extensive natural-service herds can have relevant effects in the health of bulls and negative consequences in their productive performance. Recent progress has been made in order to elucidate the pathogenesis of this disease. In this context, the study of biomarkers of inflammation in serum would contribute to gaining knowledge about the physiopathology of bovine besnoitiosis. Serological biomarkers could help in early diagnosis and prognosis, as seropositive bulls may have mild or severe testicular lesions., we have investigated the diagnostic and/or prognostic value of a panel of serum  biomarkers related to inflammation, including total protein, globulin and albumin, haptoglobin (Hp), adenosine deaminase (ADA) paraoxonase-1 (PON-1) and acetylcholinesterase (AChE) in naturally and experimentally B. besnoiti-infected males classified according to different clinical phases of the disease .HereinResults showed a similar response pattern in these biomarkers for naturally and experimentally infected cattle, with a few relevant variations. Most significant changes occurred during the acute phase of infection, although significant changes in a few biomarkers were also observed during the chronic infection. Haptoglobin, albumin, PON-1 and ADA were identified as the biomarkers that showed changes of higher magnitude in the acute phase of the infection, whereas high total protein and globulin values were found in chronically infected cattle. We have described the changes of a panel of inflammatory biomarkers of acute and chronic bovine besnoitiosis.In summary, several biomarkers with promising diagnostic value have been identified. The biomarkers associated with acute infection are related to previously reported molecular biomarkers in testicular parenchyma of infected bulls and could help in the diagnosis of early infections and complement results from specific immunoglobulin M (IgM) detection. Besnoitia besnoiti is a chronic and debilitating disease. It is a well-known endemic cattle disease in sub-Saharan countries  according to clinical signs and serological results. Molecular results as well as macroscopic and microscopic lesions were available and are shown in Table B. besnoiti IgG (early acute infection [EA]) and (ii) sera from seven bulls with IgM and IgG levels (later acute infection [LA]).Twelve acutely infected bulls were analysed. All animals showed clinical signs characteristic of the acute phase, such as fever and orchitis. The acutely infected animals were divided into two subgroups as described in Table Nine serum samples from seropositive chronically infected bulls were analysed. These animals showed clinical signs and lesions compatible with chronic besnoitiosis, such as scrotal skin hyperkeratosis and testis atrophy with azoospermia Table .Thirty-four serum samples from subclinically infected bulls were also analysed. These included seropositive fertile bulls without clinical signs or lesions Table .All animals came from extensive natural-service herds.B. besnoiti and came from herds without a previous history of bovine besnoitiosis were included in this study -3-methylaniline and 4-aminoantipyrine. These molecules, in the presence of peroxidase, generate quinine dye, which is kinetically monitored at a 550-nm wavelength  were included in the analyses. Statistical significance for the analysis was established with P < 0.05 using GraphPad Prism 6.01 software .The levels of APPs , an oxidative stress marker (PON-1) and three additional markers of inflammation  were analysed by repeated-measures two-way analysis of variance (ANOVA) and Tukey’s post hoc tests in experimentally infected calves. The non-parametric Kruskal–Wallis test was used to compare naturally infected cattle groups, and Dunn’s post-test was used for multiple comparisons, as the data were not normally distributed. Two different analyses were performed in naturally infected bulls: (i) acutely  and chronically infected bulls with testicular degeneration and subclinically fertile bulls were compared, and (ii) bulls classified in the different categories according to clinical signs and serological results [bulls with (i) early acute infection  and 12 dpi , and concentrations decreased thereafter. In both the intravenously and intradermally inoculated groups, this increase was not significant at 8 dpi, and then the levels decreased, followed by a significant increase at 22 dpi in the intravenously inoculated group  and at 29 dpi  and 33 dpi  in the intradermally inoculated group. Next, Hp levels decreased until the end of the study.Haptoglobin concentrations were increased from 8 to 12 days post-infection (dpi) in all infected groups. This increase was significant in the subcutaneously inoculated group at 8 dpi  in the subcutaneously inoculated group; higher levels at days 33, 47, 54 and 75 dpi  = 5.94, P < 0.05) in the intravenously inoculated group; and a significant decrease at 22 dpi , followed by an increase at 33 dpi  and maintenance of higher levels at 47 and 75 dpi  = 5.94, P < 0.05) in the intradermally inoculated group.Total protein levels were similar among the three inoculated groups with the exception of a few relevant findings when compared to the control group: higher levels at 54 dpi  = 7.66, P < 0.05) and from 29 dpi in the intravenously  = 7.66, P < 0.01) and intradermally  = 7.66, P < 0.01) inoculated groups and thereafter.Globulin levels showed a significant increase compared to the control group from 22 dpi in the subcutaneously inoculated group  = 15.78, P < 0.05) until the end of the study, and in the intravenously infected group at 64 dpi  = 15.78, P < 0.01) and 75 dpi  = 15.78, P < 0.0001). The values increased similarly in both the intradermally infected group and the non-infected group.AChE values increased from 25 dpi. In general terms, in all infected groups, the levels were similar to or lower than those detected in the control group. The most relevant differences corresponded to the subcutaneously and intravenously infected group at 47 dpi, showing lower levels than the control group  = 6.53, P < 0.0001) and intravenously  = 6.53, P < 0.01) inoculated groups from 8 dpi onwards and in the intradermally inoculated group from 12 dpi onwards  = 6.53, P < 0.01).The levels of albumin were lower in all inoculated groups than in the control group. This decrease was significant in the subcutaneously  = 7.25, P < 0.01) and 19 dpi  = 7.25, P < 0.01) in the subcutaneously infected group and at 22, 26, 29 and 33 dpi  = 7.25, P < 0.05) in the intradermally infected group compared to the control group.Significantly lower PON-1 levels were observed at 15  = 3.33, P < 0.05) and to 75 dpi in the intradermally infected group  = 3.33, P < 0.05).ADA levels were generally higher in all infected groups. The most relevant differences corresponded to 19 and 33 dpi in the subcutaneously infected group  and acutely infected bulls  as shown in Fig. H-test followed by Dunn’s multiple comparison test: χ2 = 32.93, df = 2, P < 0.05 and χ2 = 18.37, df = 2, P < 0.07, respectively) and early acutely infected bulls .Total protein levels were higher in chronically infected bulls than in non-infected bulls   and early acutely infected bulls .Globulins levels were significantly higher in chronically and subclinically infected bulls than in non-infected bulls  and the negative control .Finally, the highest ADA levels corresponded to bulls with an early acute infection, followed by bulls with a late acute infection, chronically infected bulls and finally subclinically infected cattle. Significant differences were observed between bulls with an early acute infection and subclinically infected bulls . In the present study, several biomarkers showed significant variations in infected animals with a slightly different profile for naturally and experimentally infected cattle.The search for serum biomarkers can help with early diagnosis and elucidate the clinical and molecular pathogenesis puzzle of bovine besnoitiosis , 22. PreB. besnoiti infection. However, in the present study, sample limitation was unavoidable, since the time post-infection in field animals was unknown. In fact, the tissue cyst size variability observed in some animals evidenced asynchrony in the chronobiology of the infection, as previously noted for acutely infected bulls [We have reported relevant differences among different naturally infected bull groups. These animals were grouped following a restrictive criterion based on a combination of clinical, histopathological, molecular and serological findings to compare homogeneous groups according to the different clinical phases of ed bulls . Anothered bulls . MoreoveHaptoglobin, albumin, PON-1 and ADA were identified as the most promising biomarkers for the acute phase of the infection. Haptoglobin was increased, whereas albumin and PON-1 showed a decrease in experimentally infected cattle, and peaks were detected during the first month post-infection. In naturally infected cattle, the lowest values of albumin and PON-1 and the highest values of ADA corresponded to bulls with acute infection.The Hp biomarker in experimentally infected cattle appeared to be a good predictor biomarker of the infection, as the first peak of Hp levels at 10 dpi appeared simultaneously in all infected groups and coincided with the incubation period and enlarged lymph nodules just prior to fever development (between 7 and 24 dpi) , except B. besnoiti infection by Langenmayer et al. [Albumin and PON-1 peak detection varied among groups at 15 dpi in the subcutaneously inoculated group and 25 dpi in the intradermally and intravenously inoculated groups around humoral  and cellular adaptive immune responses . A significant decrease in albumin concentrations was also observed in acutely infected bulls that agrees with the decrease in serum albumin levels also reported in naturally infected bulls with acute and subacute r et al. . These fr et al.  suggester et al. . On the r et al. . Indeed,r et al. .ADA is an important enzyme that participates in neuromodulation, apoptosis, necrosis and proliferation of lymphocytes during cellular response . AccordiB. besnoiti infection.In our study, ADA levels from acutely infected bulls were higher than in the other groups of bulls . This increase in ADA activity has been described in the course of diseases that induce a cellular immune response, as for example in neosporosis  and toxoTrypanosoma evansi infections in rats [In general terms, apart from a suggested correlation of APP levels with acute besnoitiosis severity, this acute response appears to be related to a few upregulated molecular markers  detected in the testicular parenchyma of acutely infected bulls versus a downregulation of PLAT, IL-1α, IL-6, IL-8 and IL-10 in chronically and subclinically infected bulls . APPs ar in rats .B. besnoiti infection. In addition, higher antibody levels have been associated with severely chronically affected animals [High total protein and globulin values were characteristic of chronically infected cattle, in agreement with the observations made by Langenmayer et al. , 36, who animals . Moreove animals , high va animals  and chro animals . Globuli animals . In our  animals .Markers of sterility were not identified, since acutely and chronically infected sterile bulls did not show a similar pattern of acute-phase response compared to non-infected and subclinically infected animals. The most relevant finding was that AChE values were higher in late acutely infected bulls, and significantly higher levels of AChE were detected in chronically infected animals than in subclinically infected animals. An increase in AChE activity may lead to enhanced degradation of acetylcholine, a molecule that has an anti-inflammatory effect . TherefoThe levels of the different biomarkers studied in experimentally infected calves at 0 dpi and in non-infected field cattle were very similar except for PON-1, which was lower in field animals than in experimentally infected calves. Thus, PON-1 levels could be influenced by animal age. It has already been described that elderly people have lower PON-1 levels than adults and children .We have described the pattern of the acute-phase response during acute and chronic bovine besnoitiosis. The results have identified several biomarkers with promising diagnostic value. The biomarkers associated with an acute infection  are related to previously reported molecular biomarkers in the testicular parenchyma of infected bulls and could help in the diagnosis of early infections and complement IgM detection.On the other hand, the value of AChE associated with elevated globulin and total protein levels as indicative of testicular injury remains to be elucidated. The kinetics of the different biomarkers should be corroborated in future longitudinal field studies, since biomarkers could aid in decisions regarding the use of seropositive bulls for natural mating.


Rubus spp. is European red raspberry (Rubus idaeus L. subsp. idaeus). Often cultivated for its high nutritional value, the red raspberry (Rubus idaeus) is susceptible to multiple viruses that lead to yield loss. These viruses are transmitted through different mechanisms, of which one is invertebrate vectors. Aphids and nematodes are known to be vectors of specific raspberry viruses. However, there are still other potential raspberry virus vectors that are not well-studied. This review aimed to provide an overview of studies related to this topic. All the known invertebrates feeding on raspberry were summarized. Eight species of aphids and seven species of plant-parasitic nematodes were the only proven raspberry virus vectors. In addition, the eriophyid mite, Phyllocoptes gracilis, has been suggested as the natural vector of raspberry leaf blotch virus based on the current available evidence. Interactions between vector and non-vector herbivore may promote the spread of raspberry viruses. As a conclusion, there are still multiple aspects of this topic that require further studies to get a better understanding of the interactions among the viral pathogens, invertebrate vectors, and non-vectors in the raspberry agroecosystem. Eventually, this will assist in development of better pest management strategies.The estimated global production of raspberry from year 2016 to 2020 averaged 846,515 tons. The most common cultivated  Insects are the largest group of invertebrates that act as plant virus vectors. In addition, mites and nematodes are also common plant virus vectors . There aNepovirus (family Secoviridae), Tobravirus (family Virgaviridae), Cheravirus (family Secoviridae), and Sadwavirus (family Secoviridae) ) and another unassigned species in the family of Secoviridae (strawberry latent ringspot virus [SLRSV]) have also been reported [Stralarivirus” within the family of Secoviridae [Secoviridae family , cherry leafroll virus (CLRV), raspberry ringspot virus (RpRSV), tobacco ringspot virus (TRSV), tomato black ring virus (TBRV), and tomato ringspot virus (ToRSV). The remaining two other viruses are CRLV and SLRSV [X. pachtaicum, are associated with at least one of the abovementioned viruses. Xiphinema diversicaudatum is known as the vector of both ArMV and SLRSV [X. diversicaudatum, ArMV may also be transmitted by X. bakeri [Xiphinema americanum is associated with several viruses infecting Rubus. It is known as a vector of ToRSV, CLRV, and CRLV in raspberry [X. americanum, ToRSV and CLRV are economically important in raspberry cultivation, while the economic impact of CRLV is more uncertain [Xiphinema vuittenezi is the only species found transmitting TRSV in raspberry in Slovakia [Longidorus sp. nematode, where both viruses are transmitted by L. elongatus, whereas L. attenuatus is only known to transmit TBRV and L. macrosoma only transmit RpRSV [L. elongatus, a mixed infection of these viruses is often observed and usually results in raspberry leaf curl disease [The plant viruses associated with longidorid-nematodes are mainly belonging to genus reported ,33. The oviridae . Raspbere family . Among tnd SLRSV ,33,54. And SLRSV ,120. Thend SLRSV . Besides. bakeri , but the. bakeri . Xiphineaspberry ,59,122 aaspberry ,124. Amoncertain ,125. ToRncertain . CLRV inncertain . This wiit RpRSV ,45,120.  disease . DespiteSitona lineatus (Linnaeus), and the pea aphid, Acyrthosiphon pisum (Harris), on pea plants have been found to promote the spread of pea enation mosaic virus (PEMV) [S. lineatus increasing the reproduction of A. pisum causing crowding and thus encouraging individuals of A. pisum to migrate to other pea plants, indirectly promoting the spread of PEMV. In addition, S. lineatus promotes the spread of PEMV by displacing A. pisum to the areas on individual plants more susceptible to virus infection [T. urticae, a non-vector in this case, was found aiding the spread of tomato yellow leaf curl virus (TYLCV) by suppressing two flavonoids in tomato plants which deter B. tabaci (the vector of TYLCV). This encourages more B. tabaci to feed on tomato plants and increase the TYLCV transmission [Otiorhynchus sulcatus) on raspberry roots has been shown to boost the population of the large raspberry aphid, Am. idaei [Myzus persicae (Sulzer) is observed in potato crops pre-infected with a plant-parasitic nematode, Globodera pallida (Stone) [M. persicae is the most important vector [Besides the groups with known or potential virus vectors discussed above, a lot of other arthropods are feeding on raspberry, including beetles (Coleoptera), moths (Lepidoptera), flies (Diptera), and sawflies and gall wasps (Hymenoptera), in addition to several groups of true bugs (Hemiptera) . All thes (PEMV) . This isnfection . In anotsmission . Similarm. idaei . Plant-p (Stone) . This wit vector . This meAphids, mites, and nematodes are the only groups proven to be involved in raspberry virus transmission. In the effort to suppress the spread of raspberry viruses, the management of these vectors plays a vital role. Their small body size and cryptic lifestyle means that low abundances may be difficult to spot before virus symptoms appear. Also, to prevent virus transmission is more demanding than managing the vectors as ordinary pests, and there is a risk that failed control efforts can increase the virus transmission instead of reducing it . Using pAm. idaei and Am. agathonica as vectors by developing aphid resistant raspberry cultivars [Am. idaei and Am. agathonica populations have evolved into biotypes that can overcome such plant resistance (better known as resistance-breaking biotypes) [Ap. idaei or Ap. rubicola have not been developed. Chemical control of raspberry aphids with insecticides, such as organophosphates, carbamate, neonicotinoids, pyrethroids, and butenolides, have been recommended to prevent spread of viruses in raspberry [Aphid management in raspberry can be categorized into four: (1) breeding of aphid resistant cultivars, (2) chemical control, (3) biological control, and (4) other methods. Great effort has been made to overcome the threat of ultivars . Howeveriotypes) ,174. Culaspberry . Howeveraspberry ,177,178,aspberry , or are aspberry . Fewer aaspberry . To sum aspberry .Aphidius ervi Haliday) seems promising, although aphid-parasitoid interactions are affected by the resistance [Beauveria bassiana  Vuill, Burkholderia spp. and Chromobacterium subtsugae Martin et al., are recommended against Am. agathonica in the US [Lecanicillium longisporum (Petch) Zare and W.Gams, Isaria fumosorosea (Wize) Brown and Smith, and Metarhizium brunneum Petch have been found effective in managing Am. idaei populations in potted raspberry grown under glasshouse conditions [In terms of biological control, aphids in raspberry have many natural enemies, such as parasitoids, ladybeetles, lacewings, and entomopathogenic microbes ,82,181. sistance . Commercn the US . In the nditions . Other mnditions  and trapnditions , physicanditions , semiochnditions , and barnditions . These aPhyllocoptes gracilis is the only mite associated with a raspberry virus. In the 1990s, this mite was effectively managed using broad spectrum insecticides, such as systemic organophosphates and endosulfan or acaricides, such as bromopropylate. Most of these pesticides have later been banned due to environmental and health impact, but some newer acaricides, like spirodiclofen and fenpyroximate also have effect [Amblyseius andersoni (Chant), Typhlodromus pyri Scheuten, and Phytoseius macropilis (Banks), can provide a good suppression of P. gracilis population [P. gracilis population under all raspberry growth conditions. Some of these phytoseiid species are classified as food-generalists, where they also feed on pollen, fungi, and plant exudates [T. pyri has been found feeding on apple leaves and fruits as well as apple powdery mildew, even in the presence of pollen and prey [P. gracilis. Entomopathogenic fungi, such as B. bassiana and Metarhizium anisopliae (Metschn.) Sorokīn, have been assessed as potential biological control agents to provide additional control of this mite. The preliminary results of this assessment seem promising, but more studies have to be carried out to confirm the efficacy [P. gracilis populations will result in less RLBV in raspberry plantations.e effect . In addie effect ,191. Biopulation ,192. Unfexudates . For exaand prey ,195. Thiand prey . On the and prey ,194. Furefficacy . EffectiLongidorus spp. and Xiphinema spp., which are vectors of raspberry viruses [Mononchoides fortidens (Schuurmans-Stekhoven), Mononchoides longicaudatus Khera, and Mononchoides gaugleri Siddiqi, Bilgrami, and Tabassum are examples of predatory nematodes that prey on several plant-parasitic nematodes including Longidorus sp. and X. americanum [Pleurotus spp., is found to produce toxin effective against several genera of plant-parasitic nematode and toxin from Pleurotus citrinopileatus Singer is more effective than other species [In general, the control measures of nematodes can be divided into: (1) chemical approaches, (2) cultural approaches, (3) host plant resistance, and (4) biological control. In chemical approaches, fumigants, such as dichloropropane-dichloropropene, methylbromide, and dazomet, and non-fumigants, such as oxime-carbamate, organophosphate, and methylcarbamates, were recommended. This has a negative environment and health impact, therefore, many of these pesticides were banned or are in the process of being phased out in the European Union  2037/2000 and (EC) 1107/2009) . Crop ro viruses . Mononchericanum . Edible Am. idaei, Am. rubi, Am. agathonica, Ap. idaei, Ap. rubicola, M. euphorbiae, M. fragariae, and Myzus ornatus and plant-parasitic nematodes  are the only proven vectors of raspberry viruses based on the current available literature. The eriophyid mite (P. gracilis) is suggested as the natural vector of raspberry leaf blotch virus (RLBV), but further studies on the transmission mechanism is required. Even though most of the invertebrate herbivores have not been reported as virus vectors, their potential involvement in the spread of raspberry viruses should not be overlooked. Also, the interaction between these pests and their respective natural enemies, such as predators, parasitoids, and entomopathogens, should be studied to develop integrated pest management strategies in raspberry plantation that can suppress the spread of viral pathogens. These strategies should also include the use of cultivars with a high degree of resistance to viruses and/or their vectors, and growing techniques that inhibit locally important vectors. As a conclusion, there are still multiple aspects in this topic which require further studies, so to have a better understanding on the complex interactions among the host plant, viral pathogens, invertebrate vectors, and non-vectors in the raspberry agroecosystem. Eventually, this will assist in development of better strategies to minimize losses caused by raspberry pests and pathogens.Twenty-two viruses have been reported to infect raspberry. Among the invertebrate herbivores found on raspberry, the aphids (


Corvus splendens). A group of six crows were intranasally inoculated with 106.0 EID50 of H9N2 virus (A/chicken/India/07OR17/2021), and 24 h post-inoculation six naïve crows were co-housed with infected crows. Crows were observed for 14 days for any overt signs of illness. Oropharyngeal and cloacal swabs were collected up to 14 days to assess virus excretion. No apparent clinical signs were observed in either infected or in-contact crows. Virus excretion was observed only in infected birds up to 9 days post-infection (dpi) through both oropharyngeal and cloacal routes. All six infected crows seroconverted to H9N2 virus at 14 dpi, whereas all in-contact crows remained negative to H9N2 virus antibodies. No virus could be isolated from tissues viz., lung, liver, kidney, pancreas, small intestine and large intestine. Although crows became infected with the H9N2 virus, transmission of the virus was inefficient to the in-contact group. However, virus excretion through oral and cloacal swabs from infected crows suggests a potential threat for inter-species transmission, including humans. Crows, being a common synanthrope species, might have some role in influenza virus transmission to poultry and humans, which needs to be explored further.This study aimed to investigate the potential of H9N2 avian influenza virus to cause disease and intra-species transmission in house crows ( Orthomyxoviridae family and are characterized based on the combinations of their surface glycoproteins, haemagglutinin (HA) and neuraminidase (NA). So far, 16 HA and 9 NA genetically distinct subtypes have been identified in birds, while H17N10 and H18N11 subtypes have been reported only in bats ) × 6.022 × 1023 = Number of molecules/μL). Then, qRT-PCR was performed to obtain a standard curve using serial ten-fold dilution of the IVT RNA , and virViral RNA was extracted from 140 µL of infected allantoic fluid using the ‘QIAamp Viral RNA mini kit’  as described by the manufacturer, and the HA gene was amplified, as described in an earlier study . The PCR