Leishmania infantum, which is responsible for causing leishmaniasis, as well as many phleboviruses, including the human pathogenic Toscana virus. We screened sand flies collected from a single site between 2017 and 2020 for the presence of both phleboviruses and Leishmania. The sand flies were sampled with attractive carbon dioxide traps and CDC light traps between May and October. We collected more than 50,000 sand flies; 2826 were identified at the species level as Phlebotomus perfiliewi (98%) or Phlebotomus perniciosus (2%). A total of 16,789 sand flies were tested in 355 pools, and phleboviruses were found in 61 pools . Leishmania was found in 75 pools and both microorganisms were detected in 16 pools. We isolated nine phleboviruses from another 2960 sand flies (five Ponticelli viruses and for Fermo viruses), not tested for Leishmania; the complete genome of a Fermo virus isolate was sequenced. The simultaneous detection in space and time of the Fermo virus and L. infantum is evidence that supports the co-circulation of both microorganisms in the same location and partial overlap of their cycles. A detailed characterization of the epidemiology of these microorganisms will support measures to limit their transmission.Sand flies transmit  Phenuiviridae, genus: Phlebovirus) are ssRNA viruses characterized by a tri-segmented genome. The L segment (6.4 kb) codes the RNA-dependent RNA polymerase (RdRp), the M segment (3.2 kb) encodes for a polyprotein that is the origin of several proteins, and the S segment (1.7 kb) encodes for two proteins with an ambisense strategy . Species in the genus were previously defined by the degree of serological cross-reactivity, but the frequent isolation of new viruses and the reassorting capacities of these viruses suggested a need to change the classification standards [Phleboviruses , is the etiological agent of neuroinvasive diseases including meningitis and meningoencephalitis. The TOSV was first isolated in 1971  and lateSeveral of these viruses had been detected in Northern Italy. In addition to TOSV, three phleboviruses—Ponticelli I virus, Ponticelli II virus, and Ponticelli III virus—were isolated . These tLeishmania genus  contribute significantly to human diseases in Italy and elsewhere. The species circulating in the Mediterranean region include L. donovani and L. infantum, which cause visceral leishmaniasis (VL), and L. tropica and L. major, which, together with dermotropic L. infantum strains, cause cutaneous leishmaniasis (CL). The estimated annual incidence of leishmaniasis in this area is 1200–2000 cases of VL and 239,500–393,600 cases of CL [Among the various sand fly-borne pathogens, the intracellular protozoa of the es of CL .Leishmania infantum is almost the only causal agent of leishmaniasis in Italy, where it causes VL and CL in humans, as well as canine leishmaniasis (canL) in dogs. Dogs are regarded as the main source of Leishmania infection in many epidemiological settings, but other domestic and wild animal species have also been implicated as reservoirs [servoirs .Leishmania strains compared to other strains [VL and CL are endemic to the hilly part of Emilia-Romagna. The first outbreak of 60 cases of VL was recorded in 1971–1972 , and a r strains ,14.Leishmania share the same principal vectors: sand flies. Sand flies are tiny hematophagous insects that often live close to domestic animals and humans. Only females bite, typically from dusk to dawn. They are poor fliers, flying silently for short distances, and they have activity peaks during summertime in non-tropical regions; sand flies are widely distributed in Mediterranean countries. The two species that are most abundant in the natural and rural environments of the Emilia-Romagna region are Ph. perniciosus and Ph. perfiliewi; both species are proven vectors of Leishmania infantum and TOSV in Italy [Phleboviruses and in Italy .Leishmania parasites in sand flies collected at a site in the Valsamoggia municipality (BO) in the Emilia-Romagna region, Italy from 2017 to 2020.In this work, we report the contemporaneous detection of diverse phleboviruses and Between 2017 and 2020, we sampled a single site that had already been surveyed, beginning in 2012  . This si2), and standard miniature CDC light traps. One carbon dioxide trap was used at the same site for every sampling and was used as an estimate of the abundance of sand flies. Other traps were employed in variable numbers, from one to nine, to maximize the number of insects collected, keeping a minimum distance of 20 m between them to avoid reciprocal interference , Dext3r (https://simc.arpae.it/dext3r/ accessed on 10 April 2021). The average of the precipitation recorded at four stations  was used, and average temperatures were recorded at the Guiglia station.Weather data from the closest climatic stations were retrieved with the web app of the Regional Environmental Protection Agency (https://blast.ncbi.nlm.nih.gov/Blast.cgi accessed on 10 April 2021). We detected Leishmania parasites with real-time PCR targeting the kinetoplast DNA [Leishmania were calculated for every sample, assuming the presence of at least one Leishmania-positive sand fly per positive pool. Primers of the PCRs are reported in We examined pools of 50 females for biomolecular analysis. The pools were ground, and the genetic material was extracted with an automated extractor  and retro-transcribed. We searched phleboviruses with PCR (henceforth pan-phlebo-PCR), targeting a 370-nucleotide region of the S segment . The ampA Fermo virus isolate was sequenced on a MiSeq Instrument  as described elsewhere . The seqg for 15 min. An aliquot of these homogenates was submitted to pan-phlebo-PCR. The samples were inoculated in a confluent monolayer of VERO cells (African green monkey kidney cells) at passage 170  and incubated at 37 °C with 5% CO2. We observed the cultures daily for 7 days to detect and characterize cytopathic effect (CPE). Then, we sub-cultured the cryolysates twice into fresh monolayers. Pools that were utilized for virus isolation were not tested for the presence of Leishmania.A sub-sample of the females was grouped in 25-specimen pools on a surface kept at −80 °C, and tested for isolation as quickly as possible, as reported previously . The insPh. perfiliewi (98%) and 45 were identified as Ph. perniciosus (2%) , and morphologically identified 2826 specimens: 2622 were identified as sus (2%) . The numer years , with a er years .Leishmania through the PCR, and 61 produced a sequence ascribable to a phlebovirus: 6 Toscana viruses, 42 Fermo viruses, 7 Ponticelli viruses, 2 Corfou viruses, and 4 of a putative phlebovirus that had been detected in the surveyed area  .We tested 16,789 sandflies —grouped yed area . The paru virus) . Using oLeishmania recorded in different seasons are shown in Leishmania circulation .The MIRs of culation , and thePh. perfiliewi in Iran in 1976. The maximum likelihood tree obtained with the Fermo RdRp amino acid sequence is shown in We attempted to isolate virus from 120 pools—100 in 2018 (collected from 18 June to 20 September) and 20 in 2019 (collected on 19 July)—for a total of 2960 sand flies . We detePh. perfiliewi species, which was much more abundant than Ph. perniciosus at the same site, as previously recorded in the neighboring area [Lutzomyia species in South America [Leishmaniasis was recorded in the Emilia-Romagna region after seasons with abundant captures of sand flies, such as in 2013–2014 [Sand flies were abundant at this study site, as demonstrated by the 5600 specimens collected in one trap in one night in 2017. This abundance was mainly sustained by the ing area ,24. The  America ,26. As a America . Similar013–2014 . Interes013–2014 . If thisOrthobunyavirus genus [We recorded the presence of at least four different phleboviruses at the same site during the four seasons of surveillance, similarly to the results obtained in the geographical area around the site . In addius genus ,33. The The isolates obtained confirmed the major adaptability of the Ponticelli viruses to cell cultures, highlighting their ability to displace other viruses that are present in an insect pool, as observed with the isolation of one Ponticelli virus from a pool that was positive for Fermo virus. Fermo virus also caused a weak CPE, suggesting a minor adaptability to cell culture; despite this, we were able to isolate and make available the complete genome of Fermo virus. Its relationship with the closest Tehran virus confirmed that Fermo virus represents a new phlebovirus species according to the 95% threshold in the RdRp sequence. The virus is closely related to Balkan viruses; however, in the absence of a complete sequence of the L segment, it is not possible to draw firm conclusions on their relation. The possible presence of a vertebrate host of this virus and its potential pathogenicity require additional studies.L. infantum has been recorded since 2012 [L. infantum in the canine population in the Emilia-Romagna region is low when compared to the CanL seroprevalence in other endemic areas of Italy, and it has shown a decreasing trend over time [L. infantum in the Emilia-Romagna region—one affecting dogs and the other circulating in Ph. perfiliewi and humans [Leishmaniasis is a well-known human health threat, and the surveyed site is located in an area in which the recrudescence of VL caused by nce 2012 ,28. Morence 2012 . The higver time . It is nd humans ,14,16. TL. infantum and phleboviruses has already been described in the Mediterranean basin [Leishmania, and we found a significant relationship in the co-detection of both on particular days of sampling. The simultaneous detection in space and time of the Fermo virus and Leishmania is an evidence of the cycle similarity between the two microorganisms and strongly suggests an overlap between their cycles. An obvious point of convergence was the sharing of the vector, Ph. perfiliewi, which was the most abundant sand fly species recorded and the main vector of L. infantum in this area [Leishmania sampled in that area showed a unique epidemiology, which strongly suggests the presence of a reservoir that has not yet been determined. Additional studies are required to determine if this statement can be extended to other protozoa and Fermo viruses. The possibility of its similarity to the ecology of phleboviruses can help in clarifying the cycles of both.The co-circulation of an basin ,36,37,38his area . InteresLeishmania through coinfection with phleboviruses also has been suggested [Sand flies can host a variety of microorganisms, including viruses and parasites, in strict spatial and temporal coexistence. The definition of the cycles of these microorganisms and the possible interactions between them could lead to better understanding of this complex picture. The potential ability to increase the infective capacity of uggested . The cyc


Pooled data showed no differences between ST and stretching on ROM . Sub-group analyses based on risk of bias, active vs. passive ROM, and movement-per-joint analyses showed no between-protocol differences in ROM gains. (4) Conclusions: ST and stretching were not different in their effects on ROM, but the studies were highly heterogeneous in terms of design, protocols and populations, and so further research is warranted. However, the qualitative effects of all the studies were quite homogeneous.(1) Background: Stretching is known to improve range of motion (ROM), and evidence has suggested that strength training (ST) is effective too. However, it is unclear whether its efficacy is comparable to stretching. The goal was to systematically review and meta-analyze randomized controlled trials (RCTs) assessing the effects of ST and stretching on ROM (INPLASY 10.37766/inplasy2020.9.0098). (2) Methods: Cochrane Library, EBSCO, PubMed, Scielo, Scopus, and Web of Science were consulted in October 2020 and updated in March 2021, followed by search within reference lists and expert suggestions (no constraints on language or year). Eligibility criteria: (P) Humans of any condition; (I) ST interventions; (C) stretching (O) ROM; (S) supervised RCTs. (3) Results: Eleven articles ( Joint range of motion (ROM) is the angle by which a joint moves from its resting position to the extremities of its motion in any given direction . ImproviIt should be noted that muscle weakness is associated with diminished ROM ,29,30. SNevertheless, studies comparing the effects of ST and stretching in ROM have presented conflicting evidence ,44, and The methods and protocol registration were preregistered prior to conducting the review: INPLASY, no.202090098, DOI:10.37766/inplasy2020.9.0098.Articles were eligible for inclusion if published in peer-reviewed journals, with no restrictions in language or publication date. The Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines were adopted . ParticiThe study excluded reviews, letters to editors, trial registrations, proposals for protocols, editorials, book chapters, and conference abstracts. Exclusion criteria, based on P.I.C.O.S., included: (i) Research with non-human animals; (ii) non-ST protocols or ST interventions combined with other methods ; unsupervised interventions; (iii) stretching or ST + stretching interventions combined with other training methods ; protocols without stretching; unsupervised interventions; (iv) studies not reporting ROM; (v) non-randomized interventions.Six databases were used to search and retrieve the articles in early October 2020: Cochrane Library, EBSCO, PubMed (including MEDLINE), Scielo, Scopus, and Web of Science (Core Collection). Boolean operators were applied to search the article title, abstract and/or keywords:  AND (“flexibility” OR “stretching”) AND “range of motion” AND “random*”. The specificities of each search engine included: (i) Cochrane Library, items were limited to trials, including articles but excluding protocols, reviews, editorials and similar publications; (ii) EBSCO, the search was limited to articles in scientific, peer-reviewed journals (iii) PubMed, the search was limited to title or abstract; publications were limited to RCTs and clinical trials, excluding books and documents, meta-analyses, reviews and systematic reviews; (iv) in Scielo, Scopus and Web of Science, the publication type was limited to article; and (v) Web of Science, “topic” is the term used to refer to title, abstract and keywords.An additional search was conducted within the reference lists of the included records. The list of articles and inclusion criteria were then sent to four experts to suggest additional references. The search strategy and consulted databases were not provided in this process to avoid biasing the experts’ searches. More detailed information is available as Updated searches: on 8 March 2021, we conducted new searches in the databases. However, each database has specific approaches to filtering the searches by date. In Cochrane, we searched for articles entering the database in the previous 6 months. In EBSCO, we searched for all fields starting from October 2020 onwards. In PubMed, the entry date was set to 1 October 2020, onwards. In Scielo, Scopus and Web of Science, publication date was limited to 2020 and 2021.Here, we provide the specific example of search conducted in PubMed: AND (“flexibility” [Title/Abstract] OR “stretching” [Title/Abstract])) AND (“range of motion” [Title/Abstract])) AND (“random*” [Title/Abstract]).After this search, the filters RCT and Clinical Trial were applied.J.A. and F.M.C. each conducted the initial search and selection stages independently, and then compared result to ensure accuracy. J.F. and T.R. independently reviewed the process to detect potential errors. When necessary, re-analysis was conducted until a consensus was achieved.J.A., F.M.C., A.A.M. and J.F. extracted the data, while J.M., T.R., R.Z. and A.M. independently revised the process. Data for the meta-analysis were extracted by JA and independently verified by A.A.M. and R.R.C. Data were available for sharing.a posteriori; (iii) ROM testing: joints and actions, body positions , mode of testing , pre-testing warm-up, timing , results considered for a given test , data reliability, number of testers and instructions provided during testing; (iv) Outcomes: changes in ROM for intervention and comparator groups; (vi) funding and conflicts of interest.Data items: (i) Population: subjects, health status, sex/gender, age, training status, selection of subjects; (ii) intervention and comparators: Study length in weeks, weekly frequency of the sessions, weekly training volume in minutes, session duration in minutes, number of exercises per session, number of sets and repetitions per exercise, load , full versus partial ROM, supervision ratio; in the comparators, modality of stretching applied was also considered; adherence rates were considered The risk of bias (RoB) in individual studies was assessed using the Cochrane risk-of-bias tool for randomized trials (RoB 2) . J.A. ang effect size (ES) [Meta-analysis was conducted when ≥3 studies were available . Pre- anize (ES) . When meize (ES) . When ROize (ES) .2 statistic, with values of <25%, 25–75%, and >75% considered to represent low, moderate, and high levels of heterogeneity, respectively [The inverse variance random-effects model for meta-analyses ,54 was uectively . Data usp < 0.05 implying bias. To adjust for publication bias, a sensitivity analysis was conducted using the trim and fill method [Publication bias was explored using the extended Egger’s test , with p l method , with L0l method .Using a random-effects model and independent computed single factor analysis, potential sources of heterogeneity likely to influence the effects of training interventions were selected, including (i) ROM type , (ii) studies RoB in randomization, and (iii) studies RoB in measurement of the outcome . These ap ≤ 0.05. Data for the meta-analysis were extracted by JA and independently verified by A.A.M. and R.R.C.All analyses were carried out using the Comprehensive Meta-Analysis program . Statistical significance was set at a priori. Directness was guaranteed by design, as no surrogates were used for any of the pre-defined P.I.C.O. dimensions. Imprecision was assessed on the basis of 95% CIs.Although not planned in the registered protocol, we decided to abide by the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) , which aAn initial search returned 194 results . After removal of duplicates, 121 records remained. Screening the titles and abstracts for eligibility criteria resulted in the exclusion of 106 articles: 26 were not original research articles , 24 were out of scope, 48 did not have the required intervention or comparators, five did not assess ROM, two were non-randomized and one was unsupervised. Fifteen articles were eligible for full-text analysis. One article did not have the required intervention , and twoUpdated searches: in the renewed searches, 28 records emerged, of which two passed the screening. However, these two records had already been included in our final sample. Therefore, no new article was included.A manual search within the reference lists of the included articles revealed five additional potentially fitting articles. Two lacked the intervention group required ,75, and The data items can be found in Interventions lasted between five  and 16 wHip joint ROM was assessed in seven articles ,72,73,78In seven articles ,72,78,85Comparisons were performed between ST and stretching groups, involving eleven articles and 452 participants. Global effects on ROM were achieved pooling data from the different joints. One article did not have the data required , but theFrom the five articles, including both genders, four provided pooled data, with no distinction between genders ,44,46,71p = 0.206; I2 = 65.4%; Egger’s test p = 0.563; Effects of ST versus stretching on ROM: no significant difference was noted between ST and stretching  was found when programs with high RoB  were compared to programs with low RoB  ( = 0.0%) .Effects of ST versus stretching on ROM, moderated by study RoB in measurement of the outcome: No significant sub-group difference in ROM changes (p = 0.320) was found when programs with high RoB  were compared to programs with low RoB  (= 77.3%) .Effects of ST versus stretching on ROM, moderated by ROM type (active vs. passive): No significant sub-group difference in ROM changes (p = 0.642) was found after training programs that assessed active  compared to passive ROM  (= 15.3%) .Effects of ST versus stretching on hip flexion ROM: Seven studies provided data for hip flexion ROM (pooled n = 294). There was no significant difference between ST and stretching interventions  was found when programs with high RoB in randomization  were compared to programs with low RoB in randomization  ( = 0.0%) .Effects of ST versus stretching on hip flexion ROM, moderated by ROM type (active vs. passive): No significant sub-group difference in hip flexion ROM changes (p = 0.466) was found after the programs assessed active  compared to passive ROM  (= 56.5%) .Effects of ST versus stretching on knee extension ROM: Four studies provided data for knee extension ROM (pooled n = 223). There was no significant difference between ST and stretching interventions , with all ST versus stretching comparisons remaining non-significant.One article behaved as an outlier in all comparisons, favoring stretching , but aftThe aim of this systematic review and meta-analysis was to compare the effects of supervised and randomized ST compared to stretching protocols on ROM, in participants of any health and training status. Qualitative synthesis showed that ST and stretching interventions were not statistically different in improving ROM. However, the studies were highly heterogeneous with regard to the nature of the interventions and moderator variables, such as gender, health, or training status. This had been reported in the original manuscripts as well. A meta-analysis, including 11 articles and 452 participants, showed that ST and stretching interventions were not statistically different in active and passive ROM changes, regardless of RoB in the randomization process, or in measurement of the outcome. RoB was low for deviations from intended interventions, missing outcome data, and selection of the reported results. No publication bias was detected.High heterogeneity is expected in continuous variables , such asMost studies showed ROM gains in ST and stretching interventions, but in two studies, neither group showed improvements ,45. AlthGlobally, however, both ST and stretching were effective in improving ROM. We asked what the reason for ST to improve ROM in a manner that is not statistically distinguishable from stretching? A first thought might be to speculate that perhaps the original studies used sub-threshold stretching intensities and/or durations. However, the hypothesis that ST has intrinsic merit for improving ROM should also be considered. ST with an eccentric focus demands the muscles to produce force on elongated positions, and a meta-analysis showed limited-to-moderate evidence that eccentric ST is associated with increases in fascicle length . LikewisOne article showed significant reductions in pain associated with increases in strength . Therefoa priori. There is a risk of multiple subgroup analyses generating a false statistical difference, merely to the number of analyses conducted [After protocol registration, we chose to improve upon the design, namely adding two dimensions (directness and imprecision) that would provide a complete GRADE assessment. Furthermore, subgroup analyses were not planned onducted . HoweverDue to the heterogeneity of populations analysed, sub-group analysis according to sex or age group were not possible, and so it would be important to explore if these features interact with the protocols in meaningful ways. Moreover, there was a predominance of studies with women, meaning more research with men is advised. There was also a predominance of assessments of hip joint ROM, followed by knee and shoulder, with the remaining joints receiving little to no attention. In addition, dose-response relationships could not be addressed, mainly due to poor reporting. However, the qualitative findings of all the studies were very homogeneous, with statistical significance tests failing to show differences between ST and stretching protocols.Overall, ST and stretching were not statistically different in ROM improvements, both in short-term interventions , and in


Since the HuNoV is not cultivable, the study utilized feline calicivirus (FCV) ATCC VR-782, a recommended surrogate as challenge organism. The test followed modified ASTM E2197. Assessment of virus inactivation was performed using a plaque assay method. With irradiance at a UVC dose of 22.5 mJ cm−2, the study obtained 99.9 % virus reduction (3 log reduction). The results demonstrate that the UVC LED array can provide effective inactivation of HuNoV.Human norovirus (HuNoV) is a highly contagious pathogenic virus that is transmitted through contaminated food, water, high-touch surfaces and aerosols. Globally, there are an estimated 685 million infections annually due to norovirus, including 200 million affecting children under the age of 5. HuNoV causes approximately 50, 000 child deaths per year and costs an estimated USD $60 billion annually in healthcare. This study sought to determine the inactivation profile of ultraviolet subtype C (UVC) against norovirus using a UVC light-emitting diode (LED) array, KL265-50V-SM-WD. The array emitted radiation at 269 nm peak wavelength and a measured fluence of 1.25 mW cm Caliciviridae [Human norovirus (HuNoV), formerly known as Norwalk virus, is an RNA virus belonging to the family iviridae . This isiviridae , contamiiviridae , and caniviridae . Becauseiviridae , such asiviridae .Caliciviridae, feline calicivirus (FCV), is frequently used as a surrogate [Globally, there are an estimated 685 million cases of norovirus infections annually, with ~200 million of them being among children under 5 years old, leading to an estimated 50, 000 child deaths and healthcare costs estimated at USD $60 billion per year . On averurrogate , especiaurrogate .For RNA viruses, ultraviolet subtype C (UVC) induces inactivation that leads to RNA damage , the priThe inactivation of HuNoV is a major focus in water purification . This isA USB4000 photospectrometer (Ocean Optics) was used to confirm the emitted radiation peak wavelength of the UVC LED array. For UVC dose, confirmation was achieved using the X1 handheld optometer (Gigahertz-Optik). The UVC LED array tested in this study was KL265-50V-SM-WD, which is rated between 70–80 mW at 500 mA and was driven at 350 mA, yielding an expected 56–64 mW at beginning of life.The disinfection assay was carried out using Eagle’s modified medium with 2 % fetal bovine serum (FBS) as the test solution. The virus was applied on 20 mm diameter magnetic stainless steel discs, spread and dried at room temperature prior to exposure to UVC. The sample was put in the centre of the box, directly opposite and aligned to the light source, where maximum intensity is found. The distance between the LED and microbe was fixed at 7 cm.The inactivation experiments were performed in duplicate per irradiation period using feline calicivirus (FCV) ATCC VR-782 spread on stainless steel magnetic discs following a modified ASTM E2197: Standard Quantitative Disk Carrier Test Method . SpecifiTo quantify viable viral particles, the dilution plate method was followed, with Crandell–Rees feline kidney (CRFK) ATCC CCL-94 cell lines being used for growth . Viral pA=p.f.u./disc for control (no irradiation with UVC) and B=p.f.u./disc for UVC ON at a given irradiation period in seconds.where −2. At 12 s and a dose of 15 mJ cm−2, the LED array obtained a 2.70 log reduction of viable FCV virus (−2 or higher dose), a >3 log reduction of viable FCV virus was obtained. No inactivation differences at 18 and 22 s (22.5 and 27.5 mJ cm−2) of irradiation were revealed (The confirmed peak wavelength of the UVC array was 269 nm, and at 7 cm height  it obtaiCV virus . At 18 srevealed . The expet al., [254, a commonly used disinfection process in the water industry, established an inactivation rate constant for HuNoV [These results are specific to 269 nm against FCV, the commonly used HuNoV model organism. Although the spectral sensitivity of HuNoV has not been investigated, it should be expected that different wavelengths will perform differently against viral pathogens, even with the same dose . In coroet al.,  demonstror HuNoV . Such anThe findings from the current study demonstrate the potential application of KL265-50V-SM-WD UVC arrays in cruise ships and resorts, especially living and dining quarters, where there are high risks of HuNoV acquisition, which can lead to disease outbreaks . With ne−2 UVC dose was found to be sufficient to achieve a >3 log reduction against FCV. The use of UVC LEDs thus promises a reduction of virus transmission during outbreaks. These findings demonstrated that UVC LEDs could serve as an effective and rapid tool in the fight against human norovirus by preventing spread via fomites.In this study, a 22.5 mJ cmThe original laboratory report with data is available upon request.


Felis catus gammaherpesvirus-1 (FcaGHV1), a novel candidate oncogenic virus, infects cats worldwide. Whether the oropharynx is a site of virus shedding and persistence, and whether oronasal carcinomas harbor FcaGHV1 nucleic acid were investigated. In a prospective molecular epidemiological study, FcaGHV1 DNA was detected by cPCR in oropharyngeal swabs from 26/155 (16.8%) of cats. Oropharyngeal shedding was less frequently detected in kittens ≤3 months of age  than in older animals; >3 months to ≤1 year: 8/26, 30.8%, ); >1 year to ≤6 years: 10/20, 50%, ); >6 years: 3/15, 33% (p = 0.078). Provenance (shelter-owned/privately owned) was not associated with shedding. In situ hybridization (ISH) identified FcaGHV1-infected cells in salivary glandular epithelium but not in other oronasal tissues from two of three cats shedding viral DNA in the oropharynx. In a retrospective dataset of 11 oronasopharyngeal carcinomas, a single tumor tested positive for FcaGHV1 DNA by ISH, a papillary carcinoma, where scattered neoplastic cells showed discrete nuclear hybridization. These data support the oronasopharynx as a site of FcaGHV1 shedding, particularly after maternal antibodies are expected to decline. The salivary epithelium is identified as a potential site of FcaGHV1 persistence. No evidence supporting a role for FcaGHV1 in feline oronasal carcinomas was found in the examined tumours. Gammaherpesviruses are a subfamily of Herpesviridae that have co-evolved over millennia with a diverse range of mammals. The most extensively studied gammaherpesvirus, Epstein–Barr virus (EBV), is a very common, innocuous part of the human virome in all but a minority of people where EBV infection causes infectious mononucleosis, nasopharyngeal carcinoma, B-cell lymphoma and other malignancies . FactorsFelis catus gammaherpesvirus 1 (FcaGHV1) is one of several novel gammaherpesviruses recently identified in animals [ animals ,5,6,7. U animals ,11,12,13 animals , consistOn the basis of genomic similarities to human, mouse, and primate gammaherpesviruses, we hypothesized that oropharyngeal tissues are a site of FcaGHV1 persistence and shedding. EBV, for example, is capable of infecting a variety of cell types, but epithelial cells are a major cellular target, and virus shed in the saliva of healthy carriers supports a role for epithelial cells in the amplification of virus during persistent infection . EpithelIf the oronasal cavity is a site of persistence, oncogenesis is a potential outcome. Long-term exposure of the oronasal mucosa to EBV is proposed as a risk factor for EBV-associated nasopharyngeal carcinoma (NPC)  and lympSamples were obtained at routine unrestricted necropsy with written owner consent or were residual diagnostic samples used with consent from the shelter.Oropharyngeal (OP) samples were obtained from shelter-housed cats using sterile swabs during a routine examination at shelters in California. Swab tips were placed in 100 µL of Zymo DNA/RNA Shield  and stored at −80 °C. OP swab samples were similarly obtained from privately-owned cats submitted for routine necropsy at UC Davis School of Veterinary Medicine. In addition, for each necropsy case, the following tissues were formalin-fixed and paraffin-embedded (FFPE): tongue, tonsil, labial oral mucosal, salivary gland, pharyngeal mucosa, nasal epithelium/conchae. Age, either documented or estimated, was recorded for each cat. Historic FFPE biopsy material from carcinomas of the feline oronasal cavity was identified and retrieved from the histopathology database at UC Davis.® Blood & Tissue Kit, QIAGEN, Hilden, Germany). DNA samples testing negative for feline glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using conventional PCR (cPCR) were excluded [DNA was extracted from swabs immediately after thawing using the QiaAMP DNA Micro Kit . DNA was extracted from frozen tissues and from 25 µm scrolls of FFPE tissue after being deparaffinized in xylene and rehydrated with 100%, 90% and 70% ethanol, using a commercial kit (DNeasyexcluded . A cPCR excluded .The 25-µL reactions contained HotStarTaq Plus Master Mix (Qiagen), 2.5 µL of extracted DNA, 0.2 µM concentrations for each primer GH-3F 5′-TGACATGTAACGCAGTCTATG-3′ and GH-3R 5′-TCTGTGCATGATTCGTTCCAT-3′. The mixtures were amplified with an initial denaturation at 95 °C for 5 min followed by 40 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 30 s. There was a final extension at 72 °C for 10 min.FFPE OP tissues from three cases testing positive for FcaGHV1 DNA on OP swabs, and three testing negative, were processed for ISH, as described previously  . Brieflyp ≤ 0.05.Statistical analysis was performed using IBM SPSS Statistics for Windows, Version 26.0 . Age was categorized as ≤3 months (Age Group 1), >3 months and ≤1 year (Age Group 2), >1 year and ≤6 years (Age Group 3), >6 years (Age Group 4). Distributions were tested, and descriptive statistics were derived for the continuous variable Age, and the categorical variables Age Group, Provenance (shelter-owned or privately-owned) and OP Swab PCR Result (positive or negative). Frequencies of OP Swab PCR Results were calculated for all Age Groups. Univariable associations between the categorical variables Age Group and Provenance with OP swab PCR Result were analysed using Fisher’s Exact Test with post-hoc pairwise Fisher’s Exact Tests with Bonferroni correction. Odds ratios were calculated to measure the associations between categorical variables. The effect of Age and Provenance combined as risk factors for a positive OP Swab Result was investigated using a multivariate model. Statistical significance was set at OP swabs were available from 155 cats with a median age of 3 months . Of these, 137 were shelter-owned , and 18 were privately-owned .p = 1.00). The frequency of OP shedding stratified by Age Group is presented in Overall, OP shedding was detected in 26/155 cats (16.8%). There was no difference in the frequency of OP shedding between shelter-owned cats (16.8%) and privately-owned cats , a single sample, a salivary adenocarcinoma, tested positive for FcaGHV1 DNA by PCR. Virus-positive cells were not identified by ISH in sections from the PCR positive case. All 11 tumours were investigated using ISH, regardless of their PCR status. Hybridization was detected in rare, scattered nuclei of individual tumor cells in a single PCR-negative case of nasopharyngeal papillary carcinoma . In addiThis study demonstrates for the first time that FcaGHV1 DNA is shed in the oropharynx of naturally infected cats. Viral shedding in oronasal and other body fluids is reported for other gammaherpesviruses including EBV, Kaposi’s sarcoma herpesvirus, MHV-68, EHV-2  and MCF We have shown that salivary epithelium is a target of FcaGHV1 infection using ISH on serial sections, whereas no other infected cell types were identified despite sampling of many oropharyngeal sites. Hence a role for salivary epithelium as a source of virus shed into saliva is possible. Given the small number of infected cells identified, we cannot rule out the presence of virus-infected cells in the other sites sampled. FcaGHV1-infected circulating B and T lymphocytes have previously been described in natural FcaGHV1 infection , but mucFcaGHV1 DNA shedding was commonly detected in our study, with one in three cats over 3 months of age testing positive for virus DNA on OP swabs. Although not directly comparable, a previous study of cats from the same region reported 14/50 (28%) tested positive for FcaGHV1 DNA in blood using virus-specific qPCR [An age-related pattern of OP shedding was observed, where the odds of virus detection were around 8 and 18 times higher in cats up to one year-, and up to 6 years- of age respectively, than in kittens up to 3 months of age. The small sample of cats over 6 years old likely precluded the demonstration of a significantly higher shedding frequency in that age group compared to kittens. The observed pattern of OP shedding is consistent with perinatal primary infection by FcaGHV1 being a common infection route, and an onset of virus shedding around the time that maternal antibodies are expected to wane from 6 to 16 weeks . For EBVNo evidence to support an association between FcaGHV1 and oronasal carcinomas was found. In EBV associated nasopharyngeal carcinoma, there is widespread hybridization to the EBV genome in the nuclei of neoplastic epithelial cells . The con


Placing all clients with a positive diagnosis for HIV on antiretroviral therapy (ART) has cost implications both for patients and health systems, which could, in turn, affect feasibility, sustainability and uptake of new services. Patient‐incurred costs are recognized barriers to healthcare access. Differentiated service delivery (DSD) models in general and community‐based care in particular, could reduce these costs. We aimed to assess patient‐incurred costs of a community‐based DSD intervention (clubs) compared to clinic‐based care in the Shinyanga region, Tanzania.Cross‐sectional survey among stable ART patients . For each group, we collected socio‐demographic, income and expenditure data between May and August 2019. We estimated direct and indirect patient‐incurred costs. Direct costs included out‐of‐pocket expenditures. Indirect costs included income loss due to time spent during transport, accessing services and off work during illness. Cost drivers were assessed in multivariate regression models.p < 0.001) for direct costs, 20.9 versus 8.23 (p < 0.001) for indirect costs and 32.2 versus 12.4 (p < 0.001) for total costs. Time spent accessing care and time spent in illness (hours/year) were 38.3 versus 13.8 (p < 0.001) and 16.0 versus 6.69 (p < 0.001) respectively. The main cost drivers included transportation (clinic vs. club: 67.7% vs. 44.1%) for direct costs and income loss due to time spent accessing care (clinic vs. club: 60.4% vs. 56.7%) for indirect costs. Factors associated with higher total costs among patients attending clinic services were higher education level  20.9 [5.47 to 36.3]) and formal employment (44.2 [20.0 to 68.5). Differences in mean total costs remained significantly higher with formal employment, rural residence, in addition to more frequent visits among clinic participants. The percentage of households classified as having had catastrophic expenditures in the last year was low but significantly higher among clinic participants .Overall, costs were significantly higher among clinic participants. Costs (USD) per year for clinic versus club were as follows: 11.7 versus 4.17 (Costs incurred by patients accessing DSD in the community are significantly lower compared to those accessing standard clinic‐based care. DSD models could improve access, especially in resource‐limited settings. In sub‐Saharan Africa, ART services are offered free to patients at the point of care. However, patients incur costs while accessing care. These patient‐incurred costs during care‐seeking include transportation, food, temporary caregiver costs, income loss during appointments, sometimes user fees or costs of other medications than ART. These are recognized as barriers to healthcare access , 2, 3. ADifferentiated service delivery (DSD) is a patient‐centred strategy currently promoted for HIV services . DSD entIn Tanzania, with a generalized HIV epidemic, DSD is recommended in the current HIV care and treatment guidelines as a patient‐centred strategy to expand access . For staTherefore, to fill this gap, this study aimed to determine the patient‐incurred cost incurred by stable ART patients both accessing clinic‐based care and DSD in adherence clubs in Shinyanga, a largely rural region in Tanzania.2We conducted a cross‐sectional survey nested within the Test & Treat (TnT) implementation project, details of which have been published elsewhere . BrieflyStable patients were eligible to participate in our study if they were 18 years of age or above and had been accessing services at the study sites for at least one year and/or had received follow‐up care in the DSD clubs for at least six months before the beginning of the study. Stable patients were identified according to the Tanzanian HIV care and treatment guidelines as those on first‐line anti‐retrovirals for at least six months, have not had any adverse drug reactions that require regular monitoring and no current illnesses (opportunistic infections and/or comorbidities) . They haParticipants were recruited among eligible patients attending clinic appointments or club meetings. In the clinics, a random list of participants was generated of all eligible patients with scheduled clinic appointments during the study period. In the clubs, all members were stable and offered participation in the study. After their consultation, those patients approached and providing informed consent in their preferred language were then asked to answer the study questionnaire. The study questionnaire was an adaptation of existing cost survey tools , 25. The2.1We compared the two population samples (clinic‐ and club‐based) using descriptive statistics in terms of sex, age, education, marital status, occupation, location, SES, years on ART, number of visits and insurance. To assess SES, we used the Tanzania DHC 2015 equity tool and collected data on asset ownership to calculate an asset‐based wealth index  . The indPatient‐incurred costs were categorized into direct and indirect costs. Direct costs included medical and non‐medical costs. Direct medical costs were derived by adding all OOP expenditures for additional medicines, laboratory tests or other consultations not provided as part of the free HIV service . Direct non‐medical costs included transportation costs, expenses incurred while accessing care such as food, accommodation, dietary supplements and the costs of temporary caregivers. Indirect costs included access‐ and illness‐related costs. These were estimated from the reported time spent accessing care or while unable to work multiplied by the estimated individual income. Income was derived from the questionnaire (reported monthly income) and we assumed an average of 8 hours/workday for 24 days per month to derive income per hour. Total patient‐incurred costs associated with HIV care were calculated by summing direct  and indirect (access‐ and illness‐related) costs. Costs afforded through coping strategies such as borrowing money and selling fixed assets were reported separately to describe coping strategies available in this population. Catastrophic expenditure was defined as patient‐incurred medical costs exceeding 10%  of reported household income , 29.Predictors of costs were identified using quantile (median) regression. The final regression model included all covariates. We used the Oaxaca decomposition method to quantify the difference in total costs between clinic and club attributable to participant characteristics . The dec3Of a total of 667 clients invited, 641 consented to participate (96%). The most frequent reason for non‐participation was the lack of time for an interview due to other scheduled engagements. Characteristics of participants are presented in Table 3.1All costs incurred by patients per visit/per year are presented in Table p = 0.84). Most of the costs (82.9%) were incurred while seeking care from providers outside the CTCs involved in the TnT project. Direct non‐medical costs contributed the most (73.6% and 45.3%) to total direct costs with travel costs ($20.6 and 8.91 in clinics and clubs respectively) being significantly higher among clinic participants (p < 0.001). Expenditure on food and temporary caregiver was reported rarely among participants, whereas accommodation cost was reported by one clinic participant alone.On average, direct medical costs incurred by participants per year did not differ significantly between the two settings . The main contributor to indirect costs was income loss, that is income foregone while seeking care. Only 5% among unemployed participants would have been engaged in leisure activities alone if they had no clinic visit/club meeting. On average, the time spent accessing care, that is traveling, and waiting to receive service, was significantly higher for clinic patients. The mean total travel time spent to service location per year was three times longer for clinic participants . Similarly, the mean total waiting time was twice as long  in these settings. Likewise, the mean total time spent in ill‐health per year was considerably higher among clinic participants though not significant . This is partially a reflection of different health service utilization patterns (see Table p < 0.001) and associated costs were larger among clinic participants. Most patients attended clinics or clubs on scheduled visits (>91%). However, there were more patients on scheduled visits  and fewer missed appointments  among club participants compared to clinic participants.Indirect costs per year were significantly higher among clinic participants  by service delivery models controlling for all covariates – see Tables The proportion of participants that were primary income earners per household, their average income or the average household income were similar across settings. Opportunity costs due to care access were, however, significantly higher among clinic participants. Catastrophic expenditure was significantly higher among clinic participants compared to club patients (see Table p = 0.861) participants. Coping strategies implemented by participants and their households to deal with medical expenditure and income loss were similar between groups. The majority of participants did not have any insurance in clinics and clubs (7.95% vs. 8.37%) and among those who did the most common form was the National Health Insurance (61.3% vs. 71.4%). Other ways participants coped include borrowing, mostly from individuals (10.3% vs. 7.57%) and the sale of property (10.0% vs. 7.2%). We observed no difference in these regard between both study groups.Participants reported ways in which their lives were impacted by their illness  was the main driver. Among clinic participants, higher direct costs were associated with higher education and formal employment while among club participants, they were associated with higher SES. Higher indirect costs were associated with formal employment among clinic participants and with higher income levels for both clinic and club participants. Our study, therefore, supports evidence that club‐based care is beneficial to reducing costs and improving access to ART services, especially in rural Tanzania.GBG is currently employed by Sanofi Pasteur. Sanofi Pasteur did not provide funding for this study. All other authors declare that they have no competing interests.ONE and GBG contributed to the conceptualization and design of the study. ONE conducted the field study and data collection. ONE was responsible for data analysis with guidance from GBG. ONE, LC, DK, SH, DN and TRW were all involved in the interpretation of the results. GBG was responsible for the overall scientific management of the study. ONE wrote the initial draft of the manuscript. All authors contributed to drafts of the manuscript, read and approved the final version.Ethics approval for this research study was obtained from NIMR, that is NIMR/HQ/R.8c/Vol. I/674. Written consent was obtained from individuals who agreed to participate in the study using appropriate forms which had been approved for the same as part of the ethics application.Table S1. Bivariate analysis showing mean direct, indirect and total costs per year (2019 USD) by participants characteristics irrespective of service delivery modelTable S2. Multivariate linear regression showing differences in mean costs per year (2019 USD) between clinics and clubs by participants characteristicsTable S3. Oaxaca decompositionTable S4. Annual household income and Medical expenditure in 2019 USD, Opportunity costs per visit and catastrophic expenditure per householdTable S5. Bivariate median regression showing factors associated with direct, indirect and total costs per year (2019 USD) incurred by participantsClick here for additional data file.


West Nile virus (WNV) has never been reported from Lebanon. Yet, this country is located on the flyway of migratory birds in the Middle East region. Serological screening was conducted to assess the potential circulation of this virus. Human, horse, and chicken sera were collected from the Bekaa and North districts. Specific IgG and IgY were first screened by ELISA. Then, positive samples were confirmed by plaque reduction neutralization test (PRNT). Besides this, adult mosquitoes were collected and tested for the presence of WNV RNA using conventional RT-PCR. Sera screening revealed a seroprevalence rate reaching 1.86% among humans and 2.47% among horses. Cross-reactions revealed by ELISA suggested the circulation of flaviviruses other than WNV. None of the tested mosquitoes was positive for WNV. The observed results constitute strong evidence of local exposure of the Lebanese population to this virus and the first report of equine WNV in Lebanon. Flavivirus; Flaviviridae) is a widespread mosquito-borne virus. It is transmitted to vertebrates by the bite of a broad range of mosquito species, mainly members of the Culex genus. Birds are its natural host. When infected, they often remain asymptomatic except in the United States where significant mortality is observed [West Nile virus  is widespread throughout Africa, parts of Europe, the Middle East, West Asia, and Australia. Following its introduction in 1999 to the United States of America, WNV succeeded in establishing itself in several countries in the Western Hemisphere . OutbreaCulex populations for the presence of WNV RNA.The Middle East region is endemic for this pathogen. West Nile fever cases are regularly reported from Israel ,5 and Tu2) and Zahle  (2), north of Tripoli, a relatively underdeveloped region with considerably important farming activities. All involved participants were recruited during summer (June to September) 2013 among outpatients referred to laboratories of hospitals (three in the Bekaa region) and dispensaries (two in Akkar) for blood testing. Each consenting participant donated 5 mL of blood and filled out a questionnaire that included information on his/her travel history.The serological study involved three types of hosts: humans, horses, and chickens. Two populations were included for the serosurvey in humans. The first population consisted of residents of two adjacent districts in the Bekaa valley: West-Bekaa  . This agEquine blood was collected from June to October 2015 from farms and riding clubs in rural areas in several districts of the Bekaa region (including West Bekaa and Zahle), North region (including Akkar), and South regions . InformaBlood was also collected from chickens raised in the backyards of villagers in the Bekaa region, including the West Bekaa district.The minimal sample size calculation was conducted on EpiInfo software version 7.2.2.6) using the following parameters: an expected seroprevalence of 8% based on what is observed in neighbouring countries [.2.2.6 usCulex species mosquitoes, Staten Island, NY, 2000) and a negative antigen with uninfected cells as described [2CO3, 0.035 M NaHCO3, pH 9.6). The negative antigen was placed in each well of every third column  while the positive antigen was distributed in the wells of the remaining columns of each plate. The antigens were allowed to bind during an overnight incubation at 4 °C. The following day, the coat solution was discarded, plates washed three times with Phosphate Buffered Saline with 0.05% Tween (PBS-T), then 100 μL blocking buffer (5% skimmed milk in PBS-T) was added, and plates were left in a humid chamber at 37 °C for 1 h. Afterward, the blocking solution was discarded and plates were washed as above. A volume of 50 μL of each serum, diluted 1:200 in PBS-T with 0.5% Bovine Serum Albumin (BSA), was placed to one negative and two positive antigen-containing wells. Plates were then incubated in a humid chamber and left for 1 h at 37 °C. After washing, 50 μL of goat anti-human IgG (H+L) horseradish peroxidase (HRP) secondary antibody , diluted 1:4000 in PBS-T-BSA buffer, were added to each well. For horse and chicken sera, goat anti-equine IgG (H+L) HRP  or goat anti-chicken IgY (H+L) HRP  were respectively used at the same dilution. After incubation and washing as above, potentially formed immune complexes were revealed by the addition of 50 μL of 3,3′,5,5′-Tetramethylbenzidine (TMB) substrate  for 10 min. The reactions were stopped with 50 μL of 1:20 HCl, and the optical density (OD) of each well was read at 450 nm. For each plate blank (wells without test sera), controls (positive and negative) were included. For each serum, a ratio of the mean of OD obtained on the positive antigen (ODP) over the OD obtained on the negative antigen (ODN) was calculated. Human sera, for which OPD/ODN >2 were considered positive, were tested by seroneutralization for confirmation. For horses and chicken sera, in order to increase the sensitivity, those with ODP/ODN >1.20 were tested by seroneutralization.Sera obtained from collected blood of different hosts were tested for the presence of specific antibodies (IgG and IgY) using an in-house indirect enzyme-linked immunosorbent assay (ELISA) as described by Ebel and collaborators (2002) . Two typescribed . The opt5 cells/mL) was prepared and dispensed (3 mL/well) in 6 well plates which were then incubated at 37 °C and 5% CO2. Test sera were serially diluted from 1:5 through 1:160 in BA-1 diluent  to 100 µL. They were then mixed to 100 µL of WNV suspension (Strain 3100365) containing 200 PFU and incubated overnight at 4 °C. The following day, 100 µL of each serum-virus mixture was added onto confluent monolayers of the prepared Vero cell plates and allowed to adsorb at 37 °C for 1 h, after which an agarose overlay (3 mL/well) was added, and incubation was continued. When virus plaques became visible, a second overlay of nutrient-agar (3 mL/well) containing neutral red  was added. Plates were then returned to the incubators. Plaques were counted on the 3rd day after the viral inoculation. The titer was determined as the inverse of the highest dilution of serum neutralizing 90% of the inoculum when compared to the control (back-titration).All sera revealed positive by ELISA (at the fixed threshold) were tested by PRNT as previously described . Vero ceIn order to assess the potential cross-reactivity with other flaviviruses, a set of WNV ELISA-positive sera were tested for DENV by PRNT following the same protocol used for WNV. DENV-2 serotype (New Guinea C) was used in this assay.2 source and BG-sentinel traps (Biogents ®) with Bg-lure attractant. Collected mosquitoes were identified using regional keys [Adult mosquito specimens were collected around houses and in farms between August and September 2014 from seven locations: 4 in West Bekaa and 3 in Akkar districts . Two typnal keys ,12,13 anTotal RNA was extracted from mosquito pools using NucleoSpin RNA isolation kit . To produce cDNA, standard reverse transcription polymerase chain reaction (RT-PCR) using GeneAmp Gold RNA PCR Reagent Kit  was performed as per the manufacturer’s protocol. Subsequently, a 326 base-pairs fragment of the NS5 gene was amplified. The PCR mix included 25 µL of PCR Master Mix , WN 9794 (GGA-ACC-TGC-TGC-CAA-TCA-TAC-CAT-C), and WNV NS5 WN 9483 (CAC-CTA-CGC-CCT-AAA-CAC-TTT-CAC-C) ,15 primeA total of 1091 serum samples were analysed: 803 from the Bekaa area and 288 from Akkar. Participants’ age ranged from 9 to 85 years (Median 39) with the highest representation of those aged between 21 and 30 years old. The majority of participants (63. 34%) were females.n = 803) and 2 from North area  had ODP/ODN > 2 and therefore were considered tentatively seropositive for WNV  belonged to four other species: Culex martini, Culex perexiguus, Culiseta longiareolata, and Orthopodomyia pulcripalpis  in participants who had never travelled out of the country confirms the local circulation of this virus. Potential cases of West Nile Fever (WNF) may go unnoticed in the country probably because infectious disease specialists are not familiar with this arboviral agent. Consequently, a West Nile infection probably would be clinically classified as an unexplained viral infection. The majority of seropositive cases were from the Southern part of the Bekaa region, a rural area with several water plans (Aammiq swamps and Qaraoun lake) and stopovers for migratory birds, which makes it suitable for the introduction and local spread of WNV. This area is the geological extension of the Great Rift Valley in Israel where a cross-sectional study, involving more than 3100 healthy individuals, showed that it had the highest seroprevalence 13.5%) of WNV  of the country . In this% of WNV Unexpectedly, the observed seroprevalence rate, especially in the Bekaa region, is comparable to that reported from endemic areas such as New York (2.6%)  and nortIn Lebanon, in a previous serosurvey conducted by Gallian and collaborators (2010) among 639 blood donors, 6 were WNV seropositive by ELISA, among which only 3 (0.47%) had neutralizing antibodies positive by PRNT at titres higher than 1/10. Seropositive participants lived in South Lebanon, Beirut, and the Bekaa; however, the authors did not discuss their travel history and whether they were locally exposed to the virus .n = 315) were seropositive by ELISA [The detection of seropositive horses among local breeds is additional evidence of WNV circulation in Lebanon. It is the first record of equine exposure to WNV in Lebanon. According to the information system of the OIE , no West Nile infection has been reported in animals since 2006 not only in Lebanon but also in Syria , while cby ELISA .The absence of WNV-specific antibodies (IgY) among poultry samples from the Bekaa suggests that there was no obvious active circulation of this virus at the time of the study.Aedes aegypti, the only vector of the virus at that time. In 2003, the tiger mosquito , a secondary vector of DENV, was observed for the first time in Lebanon [Interestingly, 52 human sera were seropositive by ELISA, some of them with high OD ratio, but did not have neutralizing antibodies for WNV. This suggests the circulation in the study region of other flaviviruses closely related to WNV, such as Zika virus (ZIKV), Usutu virus (USUV), and Dengue virus (DENV). To our knowledge, the first two viruses have never been reported in Lebanon. ZIKV had not spread yet at the time of sera collection in 2013. The only report of USUV from the Middle East is from Israel where the virus was detected in mosquito pools collected from Haifa (North), and neutralizing antibodies were detected in 10.8% of horses, predominantly from the northern part of the country ,29. Rece Lebanon . Local s Lebanon . In 2012 Lebanon . The detCulex pipiens, the known primary vector of WNV. A previous experimental study on Lebanese strains of this mosquito demonstrated a high capacity to transmit WNV [Culex perexiguus, was also present but in limited numbers. This species favors enzootic (bird to bird) and epizootic (bird to horse) transmission of WNV [Cx. pipiens and Cx. perexiguus pools, respectively [Entomological data showed a high presence of smit WNV . Moreoven of WNV . In Israectively . The absectively .In conclusion, during this study, we put forward strong evidence of humans’ and horses’ exposure to WNV in Lebanon. The virus seems more prevalent, at least among humans, in the Bekaa region than in Akkar. Serological data revealed exposure to other flaviviruses including DENV. Further studies are needed to determine the identity and the extent of the spread of these viruses.


Penaeus vannamei picornavirus (PvPV), was discovered in moribund White leg shrimp (Penaeus vannamei) collected from farm ponds in China in 2015. Similar to most picornaviruses, PvPV is non-enveloped RNA virus, with a particle diameter of approximately 30 nm. The sequence of the positive single-stranded RNA genome with a length of 10,550 nts was characterized by using genome sequencing and reverse transcription PCR. The existence of PvPV related proteins was further proved by confirmation of viral amino acid sequences, using mass spectrometry analysis. Phylogenetic analysis based on the full-length genomic sequence revealed that PvPV was more closely related to the Wenzhou shrimp virus 8 than to any other dicistroviruses in the order Picornavirales. Genomic sequence conservative domain prediction analysis showed that the PvPV genome encoded a large tegument protein UL36, which was unique among the known dicistroviruses and different from other dicistroviruses. According to these molecular features, we proposed that PvPV is a new species in the family Dicistroviridae. This study reported the first whole-genome sequence of a novel and distinct picornavirus in crustaceans, PvPV, and suggests that further studies of PvPV would be helpful in understanding its evolution and potential pathogenicity, as well as in developing diagnostic techniques.Global shrimp farming is increasingly threatened by various emerging viruses. In the present study, a novel picornavirus,  Penaeus vannamei are important restrictive considerations in the shrimp farming industry in the world [P. vannamei viruses impacting global shrimp farming to date have included white spot syndrome virus (WSSV) [Penaeus vannamei nodavirus (PvNV) [Viral diseases of he world ,2. Majors (WSSV) ,4, covers (WSSV) , infectis (WSSV) , Taura ss (WSSV) , yellow s (WSSV) , infectis (WSSV)  and Decas (WSSV) ,11,12. Ts (WSSV) ,14, CMNVs (WSSV) , IHHNV [s (WSSV) , TSV [17s (WSSV) , shrimp s (WSSV) ,20, Penas (PvNV)  and IMNVs (PvNV) . FurtherPicornavirales has undergone a significant expansion in recent years, due principally to the identification of previously unknown picornaviruses discovered in vertebrates, arthropods, algae, humans, insects and plants using “next-generation” sequencing [Picornavirales have non-enveloped icosahedral virions about 30 nm in diameter, with a pseudo-T = 3 (abbreviated “p = 3”) structure [Picornavirales from other picorna-like viruses [Picornaviridae additionally include a small protein, VP4, encoded upstream of the CP module in the polyprotein [The order quencing . The memtructure . The exitructure ,27,28,29 viruses . Moreoveyprotein . The divP. vannamei, other than an insect [P. vannamei; in addition, it has also been reported to infect other penaeid shrimp species [P. vannamei and P. stylirostris in the Americas at the beginning and then spread to farming of P. vannamei in Southeast Asia, where it has been responsible for acute mortality levels among farmed penaeid shrimp in Taiwan [Taura syndrome virus (TSV) was the first characterized picorna virus infecting an invertebrate, n insect . Taura sn insect . The mai species . TSV prin Taiwan ,36.P. vannamei sampled from a farm in China in 2015. Based on the evidence obtained in the present study supporting a new virus species, we suggest a name, P. vannamei picornavirus (PvPV), for this virus.In this paper, we describe the genome sequence, morphological features and phylogenetic relationships of a novel picorna virus discovered in P. vannamei individuals  with the same disease syndrome were sampled from a shrimp farm pond in Weifang, Shandong Province, China. The hepatopancreas tissues of the sampled individuals were fixed in 2.5% glutaraldehyde solution  for transmission electron microscope observation, and the remaining parts of the diseased shrimp sample NO. 20150715007 were preserved at −80 °C for viral isolation and meta-viromic analysis. All the protocols of shrimp handling and sampling were approved by the Animal Care and Ethics Committee, Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences .On 15 July 2015, diseased juvenile P. vannamei hepatopancreas tissues were first fixed in 2.5% glutaraldehyde in 0.1 M PBS (pH 7.4) for 24 h at 4 °C. Secondly, 1% osmium tetroxide was used for 2 h for further fixing of the sample. The sample was then embedded in Spurr’s resin for preparing ultrathin sections (50 nm). The ultrathin sections and the purified viral particles were stained with uranyl acetate and lead citrate, and examined using a JEOL JEM-1200 electron microscope .The g for 15 min to gather the supernatant. The pellets were homogenized again in 10 mL TN buffer and centrifuged at 8000× g for 15 min at 4 °C. The supernatant was clarified for a second time at 10,000× g for 25 min. The final supernatant was then centrifuged at 120,000× g for 5 h . TN buffer was used to suspend the pellet.Purification of viral particles was conducted according to a previously reported protocol with a minor revision . The samTM Magnetic Kit . The enriched mRNA was fragmented into short fragments and reverse transcribed into first-strand cDNA. Second-strand cDNA was then synthesized by amplification in vitro. Next, the cDNA fragments were purified, end-repaired, added with poly(A) and ligated to Illumina sequencing adapters. Finally, the second-strand cDNA was size-selected via agarose gel electrophoresis, amplified by PCR, and sequenced.Total RNA of the viral extracts was prepared by using Trizol reagent kit  following the kit’s protocol. The quality of purified RNA was assessed and determined by using Agilent 2100 Bio-analyzer and RNase-free agarose gel electrophoresis, respectively. mRNA was enriched from the total RNA using Ribo-ZeroPvPV was submitted for a BLAST  search, and highly similar matches were included in the dataset for phylogenetic analysis. The complete genome sequence was aligned with Clustal_W as implemented in MEGA 6.0 [Trinity, a short reads assembling program, was used to finish genome assembling . The seqMEGA 6.0  using thTotal RNA was prepared from the purified virus suspension using Viral Genome RNA Extraction Kit , and the quality and quantity of the nucleic acids were measured with the NanoDrop 2000c Spectrophotometer. Then, final concentrations of 100–200 ng/µL of the viral-RNA samples were adjusted for cDNA preparation. The primers used for synthesis of the first-strand cDNA are listed in PCR amplification was conducted with TaKaRa EX Taq DNA polymerase (TaKaRa), cDNA template, dNTP and primers following the manufacturer’s protocol. The PCR was performed at 98 °C, with pre-denaturation for 10 s, followed by 35 cycles including 94 °C denaturation for 30 s, annealing for 30 s, extension at 72 °C for 30 s and final extension at 72 °C for 7 min. The annealing temperatures were set based on the requirements of different primers . The PCRTo identify the suspected protein(s) of potential viruses, SDS-PAGE (sodium dodecyl sulphate–polyacrylamide gel electrophoresis) of the purified viruses was first conducted, and then mass spectrometry analysis of the potential proteins of the purified virus was carried out. The sample was subjected to enzymatic hydrolysis subsequently according to the previous methods ,41,42. TP. vannamei. Un-enveloped virus-like particles with a diameter of ~40 nm (n = 31) were observed in the inclusions body in cytoplasm of hepatopancreatic epithelial cells  for further investigation.TEM was used to examine the ultrathin sections of the hepatopancreas tissues of the moribund al cells a. Observ tissues b. The viP. vannamei sample NO. 20150715007. A total of 37,073,944 raw reads were obtained, and then 35,246,406 clean reads were kept after quality control. These reads were de novo assembled using Trinity, and virus-related contigs were submitted to BlastX and BlastN for primary confirmation of potential viral genomes. One potential virus with the most contigs with a whole-genome sequence of 10,550 nts in length was found, and the virus was 96% identical in genomic sequence level with the previous reported Wenzhou shrimp virus 8 (GenBank Number: KX883984.1). To confirm whole sequences of PvPV, primers for Sanger sequencing were designed based on the known gene sequences . ORF1 contained three deduced conserved domains (−39), RNA helicase  and PHA03247 super family . The deduced conserved domain of ORF2 was G-patch . The ORF1 RNA segment  encoded a RdRp, RNA helicase and the large tegument protein UL36 with a total length of 2508 aa. The ORF2 RNA segment  encoded a G-patch protein with a total length of 793 aa.Analysis of the genomic structure indicated that  domains C, includP. vannamei sample NO. 20150715007, SDS-PAGE and mass spectrometry analyses were carried out , sharing a high sequence identity of 95.43%. These results of phylogenetic analysis of genome sequence indicated PvPV was a close relative of Wenzhou shrimp virus 8. In a more refined evolutionary tree based on the most related viral genomes, PvPV seems to be classified into the genus Aparavirus , Cripavirus  and Triatovirus (type species: Triatoma virus) [Dicistroviridae. Currently, only three species of dicistroviruses have been reported in crustaceans. One was TSV in P. vannamei [P. vannamei [Macrobrachium rosenbergii Taihu virus (MrTV) in crustaceans, which was confirmed to be the causative agent of the larval mortality syndrome in M. rosenbergii [PvPV, in crustaceans, having a close relationship with the Wenzhou shrimp virus 8 (WzSV-8), MCDV-1, MrTV and TSV as shown by phylogenetic analysis. However, the potential pathogenicity of the virus is still unclear and deserves further study.itecture . The ordiviridae ,47. Basevannamei ,48,49, avannamei . One wasvannamei . The othenbergii , causingenbergii . The higPvPV and WzSV-8 have previously been classified into the genus Aparavirus in the family Dicistroviridae according to the evolutionary tree based on viral genomes. However, according to the phylogenetic tree based on the deduced amino acids of RdRp genes from the most related viruses, PvPV and WzSV-8 may be re-classified into a completely new evolutionary group markedly different from the family Dicistroviridae. In this study, compared with the RdRp and whole genomes of known viruses, the RdRp of PvPV shared higher similarity (highest at 29.79% for a matching pair with Feksystermes virus) than did the genome of PvPV (highest no more than 10% matching pair with any known viruses). The result showed that the deduced amino acids sequences of RdRp were more conservative than the genome sequence in PvPV. Therefore, it can be deduced that the result of phylogenetic analysis based on RdRp would be more reliable than that based on the genome in PvPV when carrying out more refined genetic phylogeny analysis. The finding that PvPV may be categorized as a completely new evolutionary group markedly different from the family Dicistroviridae is thus more reliable and credible. Hence, PvPV should be a new species that also represents a newly proposed genus in the Picornavirales order. This proposal will be submitted to the International Committee on Taxonomy of Viruses (ICTV) for approval.PvPV genome was arranged in this typical organization. In PvPV, two separated ORFs, flanked by UTRs, were identified. The 5′-proximal ORF1 encodes RdRp, helicase and UL36 (large tegument protein), while the 3′-proximal ORF2 was deduced to encode G-patch. In all +ssRNA viruses, the RdRp gene is the most conserved gene. Evolution of RNA viruses is defined and clarified mostly based on RdRp phylogeny supplemented by phylogenies of other genes that are conserved in subsets of viruses, together with comparative analysis of genome organization [PvPV also includes RdRp and helicase. The ORF1 of PvPV does not include proteases, a cysteine protease, or BIR but includes the large tegument protein UL36. At the same time, a conserved domain database (CDD) search identified homologous proteins to the G-patch protein in ORF2, rather than the capsid protein, which is different from most described dicistroviruses. The existence of a G-patch protein instead of the capsid protein expands our awareness of the structural flexibility and diversity among dicistroviruses. The difference in genome sequence between PvPV and other dicistroviruses supports the view that PvPV belongs to a new genus. Genomic structural analysis lays the foundation for further exploration of the pathogenic mechanism of PvPV.In dicistrovirus, the RNA genome is monopartite and dicistronic with two non-overlapping ORFs separated and flanked by the untranslated regions (UTRs) . In TSV,nization . RNA helnization . All hernization . In the nization . G-patchnization ,59,60. SPvPV, a novel picornavirus in moribund white leg shrimp P. vannamei, was discovered and characterized as a novel member of the Dicistroviridae based on virus morphology, viral genome, viral mass spectrometry analysis and phylogenetic analysis. The discovery of PvPV expands the family of Dicistroviridae and may shed light on potential PvPV disease prevention and occurrence.In conclusion,


Aedes circumluteolus mosquitoes collected in Ndumu, KwaZulu-Natal, South Africa. The AR1071 Ndumu KEDV isolate bears 80.51% pairwise nucleotide identity and 93.34% amino acid identity with the prototype DakAar-D1470 strain and was co-isolated with SPOV through intracerebral inoculation of suckling mice and passage on VeroE6 cells. This historical isolate expands the known geographic and temporal range of this relatively unknown flavivirus, aiding future temporal phylogenetic calibration and diagnostic assay refinement.The mosquito-borne flavivirus, Kedougou virus (KEDV), first isolated in Senegal in 1972, is genetically related to dengue, Zika (ZIKV) and Spondweni viruses (SPOV). Serological surveillance studies in Senegal and isolation of KEDV in the Central African Republic indicate occurrence of KEDV infections in humans, but to date, no disease has been reported. Here, we assembled the coding-complete genome of a 1958 isolate of KEDV from a pool of  Flavivirus genus (family Flaviviridae) are positive-sense RNA viruses with genome sizes between 10–11 kb classified by the International Committee on Taxonomy of Viruses (ICTV) into 53 recognized species [Aedes aegypti [The  species . The wor aegypti . ContinuAedes minutus mosquitoes collected from human baited traps in Kédougou, Senegal [A. albopictus (unknown), nor does KEDV form plaques under carboxymethylcellulose [The poorly studied Kedougou virus (KEDV) was first isolated in 1972 from a pool of 67  Senegal . Robin e Senegal . It was ellulose . Subsequellulose  indicateTo examine the ecology and geographic range of KEDV, we reviewed the literature surrounding the incidence of human cases  and mosqAedes species, including Ae. aegypti [Ae. minutus mosquitoes in Kedougou from 1996.In addition to these reports of KEDV activity in humans, several isolations of KEDV from mosquitoes have been reported in Senegal and the Central African Republic . Except  aegypti   and Powassan viruses . The firequences ,18 indicAedes circumluteolus mosquitoes, collected in Ndumu, South Africa, in 1958. Ndumu is in the coastal plain of the northern KwaZulu Natal province of South Africa with hot semi-arid climate type (Köppen classification: BSh). The Ndumo game reserve (in Ndumu area) was the focus of intensive arbovirus surveillance in the 1950s and 1960s, and several viruses were detected from mosquito catches. These include the negative-sense phleboviruses Rift Valley fever virus (RVFV), Bunyamwera and Simbu viruses [We report on the isolation of KEDV from a pool of  viruses , the alp viruses ,21, Sind viruses  and Midd viruses  and the  viruses ,20.Previously, 14 historical SPOV isolates were obtained from South African mosquitoes collected between 1958 and 1960 in the Ndumu area . The mix® Viral RNA Mini Kit , followed by cDNA preparation as described previously [Viral RNA was extracted from the clarified supernatants using the QIAampeviously . Sequenchttps://www.ncbi.nlm.nih.gov/orffinder/; Accessed 26 April 2021). For phylogenetic placement within the Flavivirus genus, 40 representative polyprotein sequences were downloaded from Genbank and aligned using MAFFT . The resultant multiple sequence alignment (40 × 3852 aa) was used to construct a consensus maximum-likelihood phylogenetic tree using IQ-TREE2 (v2.1.2) using the LG+F+R6 protein substitution model as selected using the Bayesian Information Criterion in the IQ-TREE2 ModelFinder [Random hexamer and adapter sequences were removed from the basecalled fastq files using Cutadapt v1.21, resulting in 1,273,559 clean reads . Clean relFinder . The NduAll flavivirus genomes are between ~10–11 kb and usually encode a single open reading frame that is flanked by 5′ and 3′ untranslated (UTR) regions of ~100 and ~400–700 nt, respectively. The SPAdes assembled KEDV genome is 10,555 nt, and for manual validation of the KEDV genome, 1703 (0.13%) reads (1554 paired) were remapped to the reference using BWA-MEM (v0.7.13-r1126)  and inspThe Ndumu-AR1071 KEDV genome sequence has a 5′UTR of 107nt, which is almost entirely identical (99.05%) to the KEDV DakAar-D1470 strain (Genbank: NC_012533.1). We could only recover 221 nt of the 3′UTR (compared to 390 nt of DakAar-D1470), which was quite divergent to the DakAar-D1470 strain with 87.03% pairwise nucleotide identity. Overall, the complete sequence bears 80.51% pairwise nucleotide identity to the prototype DakAar-D1470 strain 10,723 nt. The 3′UTR of flaviviruses contain highly structured, nuclease resistant elements . HoweverThe predicted polyprotein of the Ndumu-AR1071 KEDV genome has a length of 3408 aa which is identical to the size previously reported (Genbank: YP_002790882.1) . PairwisFlavivirus genus. Phylogenetically, we were able to reconstruct the overall topologies of this genus; as previously reported [Kedougou virus was initially classified as a member of the dengue virus subgroup  before treported ,18, KEDVAedes spp. mosquitoes on several occasions between 1958 and 1991 .Retrospective genomic characterization of historical arbovirus isolates at the National Institute for Communicable Diseases arbovirus repository has led to discovering a mixed virus isolate containing AR1071-SPOV and AR1071-KEDV. This represents the earliest known isolation of this virus and expands the known geographical range of this poorly studied virus.Ae. aegypti in the transmission of KEDV [Ae. aegypti [Although there is no record of human disease resulting from KEDV infection to date, serological data suggest that humans have been exposed in areas where the virus activity was reported. The close genetic relatedness of Ndumu-AR1071 KEDV to flaviviruses with severe impact on public health and evidence of the potential role of  of KEDV  should n aegypti  has faci


The California Serogroup (CSG) of Orthobunyaviruses comprises several viruses capable of causing neuroinvasive disease in humans, including La Crosse (LACV), Snowshoe Hare (SSHV), Tahyna (TAHV), Jamestown Canyon (JCV), and Inkoo (INKV) viruses. Diagnosis of specific CSG viruses is complicated by the high degree of antibody cross-reactivity between them, with laboratory standards requiring a fourfold higher titer of neutralizating antibody (NAb) activity to positively identify the etiologic virus. To help elucidate NAb relationships between neuroinvasive CSG viruses, we directly compared the cross-reactivity of NAb between LACV, SSHV, TAHV, JCV, and INKV. Mice were inoculated with individual viruses and the NAb activity of plasma samples was compared by plaque reduction neutralization tests against all five viruses. Overall, the results from these studies show that the CSG viruses induced high levels of NAb against the inoculum virus, and differing amounts of cross-reactive NAb against heterologous viruses. LACV, SSHV, and INKV elicited the highest amount of cross-reactive NAb. Interestingly, a fourfold difference in NAb titer between the inoculum virus and the other CSG viruses was not always observed. Thus, NAb titers, which are the gold-standard for diagnosing the etiologic agent for viral encephalitis, may not clearly differentiate between different CSG viruses. Peribunyaviridae of the Genus Orthobunyavirus1. Several viruses within the CSG have been reported to cause neuroinvasive disease in humans, including La Crosse virus (LACV), Snowshoe hare virus (SSHV), Tahyna virus (TAHV), Jamestown Canyon virus (JCV), and Inkoo virus (INKV)5. These five viruses are found throughout most of the world with discrete, but sometimes overlapping, geographic distributions. LACV, SSHV, and JCV are all found in the USA, with SSHV and JCV extending into Canada1. TAHV is widespread throughout Europe, Asia, and Africa, and INKV is primarily found in Scandinavia, but overlaps with TAHV in Russia1. Additionally, there is overlap between the mosquito vectors and vertebrate hosts utilized by these viruses1.The California Serogroup (CSG) is a group of at least 18 antigenically and genetically related mosquito-borne viruses in the Family Serogroup classification of viruses has traditionally been a way to group similar viruses based on the cross-reactivity of the antibodies they elicit. More recently, genetic classification of viruses has become the standard as sequencing techniques become cheaper and easier. While genetic classification gives a better understanding of the relatedness of viruses, serological relationships are still important to understand because of their implications for vaccine development and diagnostics. This is especially true for the neuroinvasive CSG viruses, as a diagnosis usually relies on serology. The virus is typically not detectable in blood or cerebrospinal fluid by the time patients present with neurological symptoms, ruling out a PCR-based approach as a diagnostic tool in most cases. A better understanding of the relationships of cross-reactive antibody between the neuroinvasive CSG viruses is critical for improving diagnosis of these viruses.8. TAHV is also in the CEV complex and is more closely related to LACV and SSHV than to JCV and INKV8. Serological relationships between these viruses have been inferred from a variety of antibody and haemagglutination assays, using different strain combinations. Historically, LACV, TAHV, SSHV, and INKV were grouped together in the California Encephalitis Complex, and JCV in the Melao Complex11. However, the early studies upon which these classifications were made relied on crude serological techniques, often compared a limited number of CSG viruses, and did not have sequence information to verify the viruses used for comparisons. While these studies are informative, there has not been a study to directly compare the differences in cross-reactive antibodies induced by the clinically-relevant neuroinvasive CSG viruses.The genetic relationships of LACV, SSHV, TAHV, JCV, and INKV have previously been evaluated in several phylogenetic studies, with similar relationships found across all segments. As summarized schematically in Fig. 12, the general function of antibodies and their role in neutralization is similar between mice and humans. Additionally, we have previously shown that pathogensis of these five CSG viruses in mice is similar to what is observed in humans13. Therefore, determining the level of cross-reactivity of NAb to the CSG viruses in mice will provide insights into the human NAb response to CSG viruses.To evaluate the cross-reactivity of antibodies elicited by LACV, SSHV, TAHV, JCV, and INKV in as controlled of a system as possible, we inoculated adult mice that are resistant to CSG virus-induced neuroinvasive disease intraperitoneally (IP) with each of these five viruses. Plasma was taken from the mice four weeks after inoculation and evaluated for neutralizing antibody (NAb) against the inoculum (homologous) virus and the other four (heterologous) CSG viruses by plaque reduction neutralization test (PRNT) to compare NAb titers and cross-reactivity. While mice and humans do have differences in specific antibody subtypes and mechanisms of development14. This complex envelope protein arrangement has made it difficult to determine specific neutralizing epitopes for the CSG viruses. However, the Gc protein, and particularly the head domain of the Gc protein, has been indicated as the primary target for NAbs16. We therefore sequenced our stock viruses to determine the exact genetic relatedness between the viruses used in this study. We then compared the cross-reactivity of NAbs between viruses to a comparison of genetic identity of the two envelope proteins, Gc and Gn, with a particular focus on the Gc head domain.The orthobunyavirus spike is comprised of three heterodimers of the two envelope proteins, Gn and Gc that are arranged in a tripod-like structure on the virion surface5 PFU of LACV, SSHV, TAHV, JCV, or INKV. At 29–30 days post inoculation (dpi), mice were euthanized and blood plasma collected. None of the mice developed clinical disease. PRNT of plasma samples were performed against the homologous virus in Vero cells. The dilution that neutralized 50% of the input virus (ND50) was calculated to evaluate the level of NAb each virus elicited in the mice. All five of the CSG viruses induced high levels of homologous NAb in all mice  response to the CSG viruses, we inoculated adult mice intraperitoneally (IP) with 10We next evaluated if the differences in homologous neutralizing antibody response could be due to sex differences. While we did not specifically design this study to evaluate sex differences, we did use a mix of male and female mice. We first evaluated if there was a difference between homologous virus ND50 values between male and female mice by combining the homologous ND50s for all viruses , but there was no significant difference via t-test (p = 0.527). We next compared homologous ND50 values between males and females within each virus , where SSHV and TAHV had three males and three females each, JCV had four males and two females, and INKV had two males and four females. No significant differences were found between males and females for any of the viruses (p ≥ 0.196). While we cannot say conclusively that there is not a sex difference due to the small sample sizes, we did not observe a difference in neutralizing antibody response to the CSG viruses between male and female mice.We next evaluated the amount of cross-reaction of NAbs between the five CSG viruses by performing PRNTs of plasma samples against the four heterologous CSG viruses. The α-LACV, α-TAHV, α-JCV, and α-INKV plasma samples all neutralized homologous virus better than the heterologous viruses, but with virus and sample variation Fig. . α-LACV Surprisingly, α-SSHV plasma neutralized TAHV better than homologous SSHV, but also had high levels of cross-reactivity against LACV, JCV, and INKV Fig. . Howeverα-TAHV and α-JCV plasma had poorer cross-neutralizing antibodies compared to α-LACV plasma. α-TAHV plasma had significantly higher ND50s against TAHV than all four heterologous viruses. The highest level of cross-neutralizing antibody was against SSHV, followed by JCV then LACV Fig. , 2. OnlyOverall, α-LACV, α-SSHV, and α-INKV plasma induced the highest homologous and cross-reactive NAb titers amongst the tested CSG viruses, while α-TAHV and α-JCV plasma had the lowest cross-reactivity. For almost all plasma samples, homologous virus was neutralized better than heterologous viruses. These findings show that the CSG viruses elicit differing levels of cross-reactive antibody, depending on the α-plasma and virus tested. Generally, neutralizing antibody titers correlated with genetic relatedness, with the closest genetically related heterologous virus having higher ND50 values than the more distantly related heterologous viruses Figs. . Surpris18. Therefore, we next looked at the fold difference between homologous virus and each heterologous virus for every plasma sample to determine how often we could discriminate the inoculum virus from our known samples with a fourfold difference. Overall, the ability to discriminate CSG viruses by a fourfold difference in NAb titer differed between CSG viruses, but this cutoff was most often not reached between genetically more closely related viruses  to 57.6% (JCV and LACV) .The amino acid % identity decreased when looking specifically at the Gc head domain, which has been implicated as the main target for host neutralizing antibodiesA sequence alignment of the Gc head domain between the five CSG viruses showed variability within this region Fig. . As expe15, there were four amino acid sites that SSHV and TAHV shared with each other that differed from the other three CSG viruses  and 1% penicillin/streptomycin solution.Virus strains LACV (human 1978), SSHV (1976), TAHV (92 Bardos), JCV (61V2235), and INKV (SW AR 83–161) were previously describedhttps://arriveguidelines.org). C57BL/6 mice greater than 6 weeks of age were used and were a mix of male and female mice and were randomized for virus inoculation. Virus stocks were diluted in phosphate-buffered saline (PBS). Prior to inoculation, mice were anesthetized with isoflurane then inoculated intraperitoneally with 105 PFU of each virus in a volume of 200 μl. Mice were checked twice daily following inoculation for clinical signs. None of the mice displayed clinical signs. At 29–30 days post inoculation, mice were humanely euthanized and blood collected via cardiac puncture using heparin to prevent coaggulation. Plasma was separated via centrifugation at 5000×g for 10 min, and separated plasma collected and stored at − 80 °C until use.Mouse experiments were performed in full accordance with the National Institues of Health Guidelines under protocol 2016-061-E, which was approved by the Rocky Mountain Laboratories Animal Care and Use Committee  and complies with the ARRIVE guidelines  was added per plasma dilution. Four virus-only control samples were included per plate. The plasma dilution + virus mixtures were gently rocked by hand, then incubated at 37 °C for one hour. Media was removed from the plates of Veros, then 200 ul of the plasma dilution + virus or virus-only samples were plated per well. Plates were incubated at 37 °C for one hour to allow for virus attachment. Wells were then overlayed with 0.5 ml of MEM with 1.5% carboxymethyl cellulose (CMC), and then incubated at 37 °C. Plates with SSHV and INKV were fixed and stained at 3 dpi, and plates with LACV, TAHV, and JCV were fixed and stained at 5 dpi. Plates were fixed by filling wells with 10% formaldehyde, incubated for 1 hour, rinsed in distilled water, then stained with 0.35% crystal violet for 10–15 min, rinsed in distilled water and inverted to air dry. Plaques were counted when plates were dry.Plasma collected from the mice was used in plaque reduction neutralization tests . Vero cells were plated in 24-well plates at a density of ~ 1.3 × 10The percent neutralization was calculated for each plasma dilution by calculating the reduction in the number of plaques compared to the mean of the virus-only control wells (at least 4 wells per assay). The ND50 was calculated for each plasma sample and virus combination by plotting the percent neutralization for each plasma dilution series vs each virus and fitting an inhibition curve  in Prism  and calculating the inhibition concentration of 50% (IC50), which is equivalent to the ND50. The ND50s were then plotted for each plasma sample vs. each virus. Comparisons of ND50 values were done via one-way ANOVA, and for each set of anti-plasma samples Dunnett’s multiple comparisons test was run comparing the ND50s of the six anti-plasma samples between the homologous virus and each heterologous virus. Sex difference comparisons were done via unpaired t-test in Prism comparing homologous ND50 values for all viruses combined between male and female mice , or within each virus that had at least two of each sex . Pearson correlations were performed in Prism between the percent amino acid identities within the Gc head domain and the heterologous virus ND50s for each inoculum virus plasma.g for one minute. The remaining steps were performed according to the manufacturer’s protocol. Sequencing was performed by the Research Technologies Section of the Genomics Unit at Rocky Mountain Labs, using the following protocol. One hundred nanograms of viral RNA was ribosomal RNA-depleted using Ribo-Zero Human/Mouse Rat . The depletion method was adjusted to account for lower inputs of RNA by using 16 μl of RNA, 2 μl reaction buffer and 2 μl Ribo-Zero probes for a 20 μl reaction rather than 40 μl. Ninety microliters of magnetic capture beads were washed and resuspended in 35 μl. The depleted RNA was purified with Agencourt RNAClean beads  and eluted in the TruSeq Elute-Frag-Prime mix . NGS libraries were prepared following the TruSeq Total RNA-Seq protocol starting at the Elute-Frag-Prime step. The constructed libraries were assessed on a BioAnalyzer DNA1000 chip  and quantified using the Kapa Quantification Kit for Illumina Sequencing . Libraries were diluted to 4 nM, pooled equally, and sequenced on the Illumina MiSeq using 11 pM as template for a Nano 300 cycle chemistry run. The sequenced reads were first trimmed and filtered for adapters and low-quality bases using Cutadapt v1.1224 and FastX-Toolkit25 respectively. Next, the cleaned reads were mapped to their respective reference genomes using Bowtie2, v2.2.926, and finally, consensus sequences and variant analysis was performed using Samtools v1.827. Sequence alignments of the consensus sequences for the translated Gn, Gc, and the head domain of the Gc protein were done using the ClustalW algorithm, and percent identities were calculated using MacVector (version 17.5.4).RNA was isolated from stock viruses comprised of cell supernatants using the QIAamp viral RNA mini kit (Qiagen) following kit protocol with the following modifications. For lysis, 560 μl of AVL buffer without carrier RNA was used. Instead of adding 500 μl of Buffer AW1, 250 μl of Buffer AW1 was used, then an on-column DNase digest was performed using the Qiagen DNase I kit. A master mix was made with 70 μl RDD Buffer mixed with 10 μl DNase I per sample, then 80 μl of master mix was added to each sample column and incubated at room temp for 15 min. Following incubation, 250 μl of Buffer AW1 was added and tubes spun at 6000×