Introduction: The recent multi-nation outbreaks of human monkeypox in non-endemic areas have created an emerging public health issue. Medical students who will become future healthcare providers are directly associated with community people and can easily sensitize the general population, so it is crucial to assess their degree of knowledge and attitudes regarding recently emerging infections or pathogens. However, studies on medical students' perception of the monkeypox virus are scarce in Saudi Arabia. Therefore, the objective of this study was to assess the monkeypox virus-related knowledge and attitudes among medical students in the country.Methods: A cross-sectional study was conducted from May to July 2022 among undergraduate medical students at King Khalid University, Abha, Saudi Arabia. A systematic random sampling technique was applied to select the study participants. A self-administered questionnaire was used to gather data on sociodemographic characteristics, knowledge and attitudes toward the monkeypox virus. Descriptive statistics and Chi-square tests were performed.Results: A total of 314 medical students were recruited for this study. The findings from this study showed that the vast majority of medical students (72%) had poor knowledge about the monkeypox virus. Respondents’ age, grade point average (GPA), fathers’ education level, and training received about the monkeypox virus were significantly associated with the level of knowledge about the monkeypox virus (p < 0.05). Nearly half of the respondents (45.9%) agreed that the monkeypox virus could be transmitted to Saudi Arabia. Overall, this study showed that the awareness levels regarding the monkeypox virus were significantly higher among seniors as compared to junior students.Conclusion: The study found poor knowledge of the monkeypox virus among currently enrolled medical students in the country’s highest-ranked medical school. This finding emphasizes the urgent need to increase their knowledge because controlling outbreaks requires significant cooperation from knowledgeable and skilled healthcare providers. In many regions of the world, the incidence of morbidity, mortality, and epidemics has increased in recent years due to the reappearance and evolution of numerous animal-borne diseases . MonkeypThe incubation period of the monkeypox virus ranges from five to 21 days . The disSince 1980, when smallpox was eradicated, the monkeypox virus has been one of the most pathogenic poxviruses. However, its recent resurgence posed a threat to global health with (WHO) declaring it to be a public health emergency in July 2022. Due to its alarming global cause, combined efforts should be made to raise awareness by promoting preventative measures, such as educating healthcare workers and the general public about early detection, response activities, and rapid risk assessment . To achiSaudi Arabia reported its first confirmed case of the monkeypox virus in a foreign patient on July 14, 2022 . InformaStudy setting and durationThis study was carried out among undergraduate medical students at King Khalid University, Abha, Saudi Arabia, who were enrolled in their second, third, fourth, fifth, and sixth academic years. This public university, located in southern Saudi Arabia, comprises 29 colleges with a range of academic disciplines, including medicine, engineering, and computer science. Approximately 60,000 students are enrolled at this university (as of the 2019 academic year). The southern region of the country has a growing population, and this renowned institution meets their basic needs, such as education, and helps policymakers by exploring scientific knowledge, which makes our study area suitable for conducting research on such emerging public health issues. The study period was from May 24, 2022 to July 20, 2022.Study design, subject, and samplingAn institution-based cross-sectional study was carried out among 314 medical students to assess their knowledge and attitude about monkeypox virus. The participants were enrolled after meeting the following inclusion requirements: (i) being undergraduate medical students and (ii) Saudi citizens by birth. Students who were reluctant to participate in the study or had major illnesses or mental issues were not allowed to participate. A systematic random sampling technique was used to choose the participants, taking into account the individuals' academic year and gender. The sample size was calculated by the appliance of the single sample proportion formula , n = z2pContent of the study toolA structured, anonymous and close-ended questionnaire was developed for this study. The electronic survey tool consisted of three parts: (i) participants’ sociodemographic information, (ii) assessment of participants' knowledge of the monkeypox virus, and (iii) assessment of attitudes toward the monkeypox virus. The first part of the questionnaire includes students’ sociodemographic information such as age, gender, education year, grade point average (GPA), fathers and mothers education level, source of information about the monkeypox virus, etc. The participants' knowledge of the monkeypox virus was assessed in the following section, which included 20 questions. The questions included information about the first isolation of the monkeypox virus, where it occurred, and which part of the world is nowadays the most affected. In addition, they were questioned about the mode of transmission, symptoms, incubation period, the most severe complications, and the likelihood of transmission of the virus to Saudi Arabia. Finally, to assess attitudes toward the monkeypox virus, a set of 10 statements on a five-point Likert scale was used . These statements are intended to elicit respondents' perspectives on the potential to control the monkeypox virus globally and their attitudes toward learning more about it, travel medicine and re-emerging infectious diseases.Reliability and validity of the questionnaireTo prepare the questionnaire, the authors of this study searched for and reviewed all possible previous research on “monkeypox virus epidemiology” and “monkeypox knowledge and attitudes.” The different databases including EMBASE, Cochrane Library, and PubMed, and the websites of the CDC, WHO, and Google Scholar were searched until May 25, 2022 for published research. The most relevant articles were chosen to construct the questionnaire . The conData collection procedurePrior to data collection, ethical clearance was obtained from the Research Ethics Committee (REC) of King Khalid University, Saudi Arabia. For data collection and entry, we used Google Forms and Excel sheets. Initially, we collected the students’ email addresses from the respective department of the institution. We emailed the questionnaire to the participants’ institutional email addresses. Each participant was briefed on the purpose and objectives of the study before being asked for participation consent. The participants were also assured that their responses to the questionnaire would be kept confidential. In addition, the participants were informed that they could withdraw from the study at any time and that participation was not required for their course.Statistical analysisData were analyzed using Statistical Package for social sciences (SPSS) software . Descriptive statistics such as frequency and percentage were computed to summarize the variables of interest . A scoring system ranging from 0 to 20 points was created to assess the level of knowledge regarding the monkeypox virus. For the knowledge item (20 questions), a “1” point was given for correct responses, and a “0” point was assigned for unknown or incorrect responses. The total score was used to determine the level of knowledge classification, with a score of 11 indicating a poor level of knowledge and 11 or higher indicating a good level of knowledge. The Pearson Chi-Square test was used to assess the associations between variables. P-values less than 0.05 were regarded as statistically significant.Three hundred fourteen of the 400 medical students who were invited to take part in the study completed the questionnaire, yielding a response rate of 78.5% Table . Male stTable Regarding licensed vaccines, approximately 73.6% of respondents correctly identified the absence of monkeypox virus vaccines. A large proportion of respondents (78.4%) believed that Saudi Arabia is at risk of being affected. In contrast, a smaller proportion acknowledged that the country is currently afflicted with chicken pox, a disease that resembles the monkeypox virus (37.2%). Nearly the same number of participants answered correctly (48.2%) and incorrectly (51.9%) that fluid therapy is the most common supportive treatment for this disease.Table The attitudes of medical students regarding the monkeypox virus, emerging diseases, and travel medicine are presented in Table The clinical management of monkeypox virus outbreaks requires the close collaboration of knowledgeable healthcare professionals. An adequate understanding of the disease is required for all aspects of disease management, including case detection, immunization, and the provision of ultimate medical care. Therefore, this study aimed to investigate medical undergraduates' knowledge, awareness, and attitudes regarding the monkeypox virus. According to our knowledge, this is the first study to assess students' knowledge and attitudes regarding the potential spread of the monkeypox virus. According to the data presented in our study, most respondents were aware of the monkeypox virus epidemic. Regarding the mode of transmission, more than half of the respondents believed it was a sexually transmitted disease. In contrast, they were also aware of blood-borne transmission and maternal-fetal transmission. Some students were also unlikely to be aware of the commonly exhibited symptoms and incubation period. Less than half of the respondents were aware of fluid therapy, despite it being the most common form of supportive therapy. 72% of respondents had poor levels of knowledge, compared to 28% who had adequate levels. These results were comparable to those of a study conducted by Ibrahim et al.  to deterHarapan et al.  conducteAccording to studies, training in healthcare facilities boosts medical practitioners' confidence ,26. ApprMost general practitioners in Indonesia (98.2%) agreed that the government should provide health care workers with monkeypox virus training, and 88.8% of general practitioners in Indonesia recommended that the virus be included in Indonesia's National Medical Curriculum . SimilarOur study revealed that both sexes possessed low levels of general knowledge regarding the monkeypox virus. In addition, it was observed that the respondents preferred self-administered questionnaires. However, our study's findings contradicted those of Albarrak et al.  they obsIn addition, it was found that respondents preferred face-to-face interviews. Ibrahim et al.  came to However, this study was not free from limitations. First, because the study was cross-sectional in nature, no causal link between the dependent and independent variables could be established. Second, this study did not include the estimated sample size due to the exclusion procedures. Third, the study was conducted at only one institution; therefore, the findings cannot be generalized to the broad spectrum of the country. Regional differences in the facilities made available to students may account for possible selection bias in the study's findings. Fourth, the scales of knowledge and attitude test were not validated, as we did not perform any factor analysis to see the construct validity of the questionnaire. Lastly, cognitive and reporting bias was possible in the questionnaire, which was based on a subjective approach.In general, this study showed that medical students had inadequate knowledge and awareness about the monkeypox virus. Students’ age, GPA, fathers’ education level, and training received about the monkeypox virus were significantly associated with the level of knowledge about the monkeypox virus. Although they were optimistic about the system's ability to manage future outbreaks of the monkeypox virus in Saudi Arabia, they were unaware of the disease transmission and possessed a limited understanding of the virus. These findings emphasize the urgent need to increase their knowledge because controlling outbreaks requires significant cooperation from knowledgeable and skilled healthcare providers. Our research suggests that, in order to make it easier for students to learn about new epidemic outbreaks, such subjects be covered in educational courses, and public health training and awareness programs.


Rhabdoviridae) and included seven genes: 3′-N-P′-P-P3-M-G-L-5′. BLASTP analysis revealed that the putative L protein had the highest amino acid sequence identity (75%) with strawberry virus 2. BCRV2 was detected in Cryptomyzus galeopsidis, but efficient transmission by this aphid was not confirmed. Of note, we observed coinfection of the KB1 line with blackcurrant-associated rhabdovirus (BCaRV) by RT-PCR. This is likely the first evidence of the presence of a cyto- and a nucleorhabdovirus in a single host.A virome screen was performed on a new breeding line, KB1, of blackcurrant. Rhabdovirus-like particles were observed by electron microscopy in ultrathin sections of flower stalks, and the complete genome sequence of a novel virus, provisionally named blackcurrant rhabdovirus 2 (BCRV2), was determined and verified using high-throughput sequencing. The genomic organization of BCRV2 was characteristic of cytorhabdoviruses (family  Plant-infecting rhabdoviruses are widespread and present in many dicotyledonous plants, including gymnosperms and monocotyledonous crops  ,2. In adRibes plants [Ribes plants, with symptoms of blackcurrant reversion and gooseberry veinbanding diseases [Betanucleorhabdovirus, BCaRV) was identified in the USA in 2018 in a single asymptomatic blackcurrant plant [Varicosavirus) was identified in the transcriptome of Siberian currant (Ribes diacanthum) [To date, at least 20 viruses have been reported infecting s plants . Among tdiseases . Later, diseases . The parnt plant  and latent plant . Recentlcanthum) . The othThe typical bullet-shape virus particles are easy to recognize, and electron microscopy has long been the main source of data on the presence of rhabdoviruses in plants. However, difficulties in mechanically transferring the virus to test plants and in isolating virus particles have resulted in incomplete biological information about many recognized rhabdoviruses, and there is a lack of sequence data for many of them.The genomes of cyto- and nucleorhabdoviruses have six conserved canonical genes encoding nucleocapsid protein (N), phosphoprotein (P), putative movement protein (P3), matrix protein (M), glycoprotein (G), and large polymerase (L). Smaller additional genes may be located between the G and L genes or, in some viruses, between the N and P and/or the P and M genes ,13. MonoSuaeda salsa seeds [Currants are propagated vegetatively, but new varieties are produced by sexual crossing . Crossinsa seeds ,17 as wesa seeds , so tranIn this work, we describe, for the first time, a mixed infection of blackcurrant with a novel cytorhabdovirus and blackcurrant-associated rhabdovirus, present a genome description of the former virus, and explore potential vectors and sources of the novel virus.Plants of the new KB1 breeding line of a dessert variety of blackcurrant were provided by Holub Ltd., Czech Republic. The material showed no visible mosaic, growth malformation, or other symptoms indicating the presence of intracellular pathogens.Ultrathin cross sections were prepared from the flower stalks from three greenhouse-grown KB1 plants at the flowering stage. Tissue pieces (approx. 2 mm × 2 mm) were fixed in 0.1 M potassium phosphate buffer, pH 7.3, 5% glutaraldehyde and 4% sucrose for 24 h at 4 °C under light vacuum. Samples were post-fixed in 1% osmium tetroxide, dehydrated in ethanol and embedded in Durcupan resin (Fluka). Ultrathin sections 50 to 70 nm thin were stained with uranyl acetate and examined in a TEM JEOL 1400 at 120 kV.cox b gene with published primers [Total RNA was purified from approximately 50 mg of leaves using the GeneJET Plant RNA purification mini kit  and used for library preparation with the NEBNext Ultra II Directional RNA Library Prep Kit and Illumina sequencing with the NEBNext Poly(A) mRNA Magnetic Isolation Module . Batches of 10 seeds were surface sterilized with 5% perchlorate for 15 minutes, washed three times with distilled water and used for RNA purification. Total RNA was purified from individual aphids using TRI reagent  according to manufacturer’s recommendation. Molecular identification of aphid species was based on Sanger sequencing of the  primers  and PhirRT-PCR and RT-qPCR analyses were performed using cDNA templates prepared using RevertAid transcription kit  and SapphireAmp PCR mastermix  and HOT FIREPol EvaGreen qPCR Mix Plus , respectively. Detection primers and primers used for genome ends sequencing are presented in High-throughput sequence data were analysed in CLC Genomic Workbench 9.1.5 , and general sequence analyses were performed in Geneious 9.1.8 . The 5′ and 3′ends were obtained using the 5′ and 3′ RACE kits  and the specific primers listed in Phylogenetic analysis on N and L proteins was performed using MEGA X software with the ML method and the JTT model  and 1000https://services.healthtech.dtu.dk/service.php?NetNGlyc-1.0. accessed on 1 July 2022.N-glycosylation sites in the G and P′ proteins were predicted at Aphis shubertii and Cryptomyzus galeopsidis  were placed together in net-protected cages containing BCRV2- and BCaRV-free blackcurrant cv. Titania and cv. Öjebyn plants for three weeks. Prior to transmission assays, individual aphids from KB1 plants were RT-PCR tested with BCRV2-specific primers 3395 and 3396 and BCaRV-specific primers 3136 and 3137 , whereas the cytorhabdovirus particles were smaller, measuring 61 ± 4.3 × 225 ± 21.4 nm (n = 11)  A–C.Two nearly complete genome sequences were assembled from the HTS data. The first sequence, 14.364 nucleotides (nt) long, was 89.9% identical at the nt level to blackcurrant-associated rhabdovirus (BCaRV) .Cytorhabdovirus ribesnigri in the genus Cytorhabdovirus, family Rhabdoviridae. The second viral genome was assembled from 13,758 individual reads, resulting in a mean genome coverage of 159. This represents approximately 0.02% of the 81 million trimmed reads. The 3′ and 5′ ends were completed using the RACE approach. The complete genome of blackcurrant cytorhabdovirus is 12,882 nt in length. This is one of the smallest genomes among cytorhabdoviruses . The genome termini are complementary in a 24 nt long region, with 1 nt long overhang at the 3′ terminus. The genome contains seven genes in the order 3′-N-P′-P-P3-M-G-L-5′ . The 3′-The conserved gene junction sequences that have been shown to play an important role in transcription termination and polyadenylation of the preceding gene transcript and transcription initiation of the following transcript are 3′ AAUUCUUUU, GNU, and C(N) . This moThe phylogenetic trees based on the L and N protein amino acid (aa) sequences are topologically identical and show that BCRV2 clustered with StrV2, LNYV, Trifolium pratense virus B, and cabbage cytorhabdovirus 1, among the cytorhabdoviruses .In plant rhabdoviruses, there is a strong correlation between phylogenetic relationships and vector types . PhylogeCryptomyzus galeopsidis) and the permanent currant aphid . Aphids from both species were screened for the presence of cyto- and rhabdoviruses using virus-specific primers and RT-PCR. A. schneideri aphids (n = 8) were negative for both viruses, while five C. galeopsidis aphids were positive for BCRV2 and two were positive for BCaRV. However, the transmission experiments with C. galeopsidis (approximately 100 aphids transmitted per plant) did not result in BCRV2 infection of cv. Titania (n = 3) or cv. Öjebyn (n = 2) plants, when tested by RT-PCR 17- and 28-days post aphid transmission. Two plants of the KB1 breeding line, kept separately in a greenhouse, were naturally colonized by two aphid species: the European blackcurrant aphid . Similarly, only minor differences (up to Cq = 1) were detected between leaves at different developmental stages  with the same result (Mixed infection with two rhabdoviruses (one cyto- and one nucleorhabdovirus) in a single plant raises new questions about possible competition between the viruses. Therefore, we determined the relative titers of both viruses using NADH mRNA as a housekeeping reference. Although we detected higher BCRV2 levels than BCaRV in individual KB 1 plants using the RT-qPCR-based analyses, the differences were not statistically significant and varied widely among biological samples  [Although high-throughput sequencing is now a relatively accessible, sensitive, and inexpensive method for virus screening, it requires some skill, expertise, and specialized software that are not routinely available in every laboratory. Using the simpler RT-PCR and degenerate primers that recognize the most conserved motifs on the L gene of rhabdoviruses, we detected cytorhabdoviruses in ivy plants and Usnea lichens ,4. Usingdovirus) , so thisBlackcurrant-associated rhabdovirus (BCaRV) was previously identified in the United States in blackcurrant germplasm imported from Russia, accession Veloy . BCaRV wThe sizes of the particles of both rhabdoviruses observed in ultrathin sections were very similar. This prevented us from accurately identifying the nature of individual particles located outside the nucleus. In this case, specific labeling should be used for such differentiation. Ribes species harbor more than 30 aphid species [Aphis schneideri, C. galeopsidis, C. ribis, Hyperomyzus lactucae, and Nasonovia ribisnigri are the most common, we found only two of them on the blackcurrant plants selected in this study. Although some C. galeopsidis individuals were positive for both viruses, virus transmission was not successful with a larger number of these aphids. It should be noted that BCRV2 is phylogenetically related to cytorhabdoviruses, whose vectors are aphids. Of particular interest would be other aphid species native to Ribes sp. The G protein of BCRV2, which is essential for the attachment of the virus to predicated midgut receptors of aphid vectors [Although  species ,27, amon vectors , is veryIn mixed virus infections, there are different types of relationships between virus species, ranging from antagonism to mutualism . We founThe effects of the viruses on blackcurrant yield are not yet known. It is somewhat surprising that plants infected with the two viruses (resulting in asymptomatic infection) have been recognized as promising for further evaluation, with good flavor and fruit size characteristics. We note that infection with a similar aphid-borne cytorhabdovirus, strawberry virus 1, reduces yield per plant by 11.7–31.8%, depending on the cultivar .Further research should focus on identifying the natural BCRV2 vector by increasing the number of transmission experiments, and determining the biological significance of the virus in terms of its incidence and impact on yield.To our knowledge, this is the first documented case of mixed cyto- and nucleorhabdoviral infection in a single host.Aphis schneideri seems to be unable to replicate and transmit BCRV2 and BCaRV. While part of the population of Cryptomyzus galeopsidis was positive for both viruses, it was unable to transmit the viruses to two blackcurrant cultivars.Interestingly,


Cucumber green mottle mosaic virus (CGMMV) belongs to the Tobamovirus genus and is an important quarantine virus of cucurbit crops. Seedborne transmission is one of the principal modes for CGMMV spread, and effective early detection is helpful to prevent the occurrence of the disease. Quantitative real-time reverse-transcription PCR (RT-qPCR) is a sensitive and rapid method for detecting CGMMV nucleic acids, but it cannot distinguish between infectious and noninfectious viruses. In the present work, a propidium monoazide (PMA) assisted RT-qPCR method (PMA-RT-qPCR) was developed to rapidly distinguish infectious and inactive CGMMV. PMA is a photoactive dye that can selectively react with viral RNA released or inside inactive CGMMV virions but not viral RNA inside active virions. The formation of PMA-RNA conjugates prevents PCR amplification, leaving only infectious virions to be amplified. The primer pair cp3-1F/cp3-1R was designed based on the coat protein (cp) gene for specific amplification of CGMMV RNA by RT-qPCR. The detection limit of the RT-qPCR assay was 1.57 × 102 copies·μL−1. PMA at 120 μmol·L−1 was suitable for the selective quantification of infectious CGMMV virions. Under optimal conditions, RT-qPCR detection of heat-inactivated CGMMV resulted in Ct value differences larger than 16 between PMA-treated and non-PMA-treated groups, while Ct differences less than 0.23 were observed in the detection of infectious CGMMV. For naturally contaminated watermelon leaf, fruit and seedlot samples, infectious CGMMV were quantified in 13 out of the 22 samples, with infestation levels of 102~105 copies·g−1. Application of this assay enabled the selective detection of infectious CGMMV and facilitated the monitoring of the viral pathogen in watermelon seeds and tissues, which could be useful for avoiding the potential risks of primary inoculum sources. Cucumber green mottle mosaic virus (CGMMV), a member of the Tobamovirus genus in the Virgaviridae family, is an economically important viral pathogen of cucurbit crops worldwide. It infects cucurbit species, such as watermelon (Citrullus lanatus), melon (Cucumis melo), cucumber (C. sativus), pumpkin (Cucurbita moschata) and bottle gourd (Lagenaria siceraria), causing serious losses to their production [oduction ,2,3,4. Coduction  and has oduction ,11,12,13oduction . The glooduction ,15,16. Coduction . Owing toduction . Therefooduction ,20,21.Commonly used methods, such as enzyme-linked immunosorbent assay (ELISA), reverse-transcription polymerase chain reaction (RT-PCR), reverse-transcription loop-mediated isothermal amplification (RT-LAMP), immunocapture-RT-PCR (IC-RT-PCR) and recombinase polymerase amplification assay (RPA) have been established and used for the qualitative detection of CGMMV ,26,27,28Propidium monoazide (PMA), an azide derivative, is a membrane-impermeable dye that is only able to enter damaged or destroyed capsids. Thus, the RNA inside live virions cannot be bound by PMA, and only the RNA from inactive virions can be bound by PMA. PMA has the property of being photoreactive and intercalating into RNA upon exposure to intense light, preventing their amplification by PCR ,33. It hcp) gene sequences; (ii) optimize the PMA concentration and light exposure time; and (iii) apply the established PMA-RT-qPCR assay to detect infectious viruses in naturally contaminated watermelon leaves, fruits and seeds.The present study aimed to assess the use of PMA coupled with RT-qPCR to distinguish between infectious and inactive CGMMV viruses in seeds and plant tissues. The objectives were to: (i) design specific primers for CGMMV based on coat protein (Watermelon mosaic virus (WMV), Cucumber mosaic virus (CMV), Melon mosaic virus (MMV), Tobacco ring spot virus (TRSV), Cucurbit chlorotic yellows virus (CCYV), Zucchini yellow mosaic virus (ZYMV), Squash mosaic virus (SqMV), Melon yellow spot virus (MYSV), Melon necrotic spot virus (MNSV), Prunus necrotic ringspot virus (PNRSV), Papaya ringspot virus (PRSV), Tobacco mosaic virus (TMV) and Potato Y virus (PVY), were provided by the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences  following the manufacturer’s instructions. Then, 1% agar gel electrophoresis was performed to determine RNA integrity. RNA concentration and purity were measured by using a SmartSpec Plus spectrophotometer . RNA samples with A260/A280 ratios within the range of 1.9–2.1 were used as templates for RT-qPCR. The RNA samples were stored at −80 °C.cp gene (GenBank: KY977430.1). The virus isolates used for studying the primer specificity in this study are listed in A primer pair cp-3-1F (5′-CCGCTAGGGCTGAGATAG-3′) and cp-3-1R (5′-CTTAGAGGTGGTAGCCTCTGA-3′) was designed for specific amplification of CGMMV RNA based on sequence alignments of the http://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 11 May 2020).For cDNA synthesis, reverse transcription was performed from 2 μg of total RNA using a FastKing RT Kit . After reverse transcription, 1 μL of cDNA was used as the PCR template in a total volume of 25 μL, containing 12.5 μL 2 × Taq PCR Master Mix  and 0.5 μL of each primer. Amplification was performed in a PCR amplifier  with an initial denaturation step at 94 °C for 4 min, followed by 34 cycles at 94 °C for 30 s, 60 °C for 30 s, 72 °C for 45 s and a final extension of 72 °C for 10 min. The amplification products of CGMMV isolates were resequenced, and the identity of the sequences was checked against the NCBI database cp gene. Then, single positive plaques were picked and sequenced for comparison with the known CGMMV cp gene sequences. The concentration of the plasmid was measured using an ultra-microspectrophotometer . The copy number of plasmid was calculated according to Fronhoffs et al. (2002) [The confirmed CGMMV virus products were purified, connected to the M5 HiPer pTOPO-TA vector , transformed into DH5α cells , inoculated onto Luria-Bertani (LB) plates  containing 50 μL/mL ampicillin and then incubated overnight at 37 °C to obtain the plasmid containing the CGMMV . (2002) .8 to 1.57 × 10 copies·μL−1, which were prepared in sterile distilled water. RT-qPCR was conducted in a 20 μL reaction volume containing 1 μL plasmid DNA, 10 μL ChamQ Universal SYBR qPCR Master Mix , and 0.4 μL of each primer cp-3-1F/cp-3-1R. Amplification was performed in the real-time PCR system  under cyclic conditions as follows: 95 °C for 30 s, followed by 40 cycles at 95 °C for 10 s and 60 °C for 30 s. Cycle threshold (Ct) values were calculated automatically by ABI 7500 software . Each dilution was analyzed in ten replicates. The standard deviation (SD) of the Ct values was determined based on ten replicates, and the coefficient of variation (CV) was calculated with the SD and the mean values of Ct (CV = 100 × SD/mean). The detection limit of RT-qPCR was specified as the lowest concentration at which replicates showed CV ≤ 35% at the calculated concentration [A standard curve for the quantification of CGMMV was generated by analyzing a 10-fold dilution series of plasmid with known quantities from 1.57 × 10ntration ,44.v/v) for 2–3 min at room temperature in a vortex oscillator. The aqueous layer containing the virus was separated from the organic phase by centrifugation at 900 g. The virus was then subjected to one or two more cycles of differential centrifugation in 0.1 M phosphate at pH 7.0, taking up the pellets in water. The obtained virus suspensions of CGMMV were then divided and prepared into two groups: infectious virus (not treated) and inactive virus (heat-killed in a hot bath at 100 °C for 20 min).The CGMMV virus suspensions were prepared according to the method described by Jones et al. (1986) and Zhang et al. (2016) ,46. Brie−1, which was stored at −20 °C in the dark. Briefly, 200 μL of either infectious or inactive virus suspension of CGMMV was treated with PMA at different final concentrations of 0, 10, 40, 80, 120, 160 and 200 μmol·L−1 in Eppendorf tubes. The tubes were first incubated for 30 min at room temperature in the dark, which allowed PMA to penetrate the inactive virus and bind to the RNA. The tubes were then placed in an ice bath with lids removed and exposed to light from a 50 W LED lamp for 15 min at a distance of 15 cm from the sample tube to activate PMA in inactive virus and to photolyze free PMA in solution. After PMA treatment, CGMMV RNA was extracted as described above for amplification by RT-qPCR. The optimal PMA concentration was determined by calculating and comparing the dCt and ddCt of PMA-treated or PMA-untreated samples of infectious or inactive viruses, with ddCt = dCt (inactive) − dCt (infectious), dCt = Ct (+PMA) − Ct (−PMA) [The appropriate concentration of PMA would be an important factor to effectively discriminate between infectious virus and inactive virus, which should inhibit the amplification of inactive virus and have no effect on the amplification of infectious virus. PMA  was dissolved in 20% DMSO to create a stock concentration of 20 mmol·Lt (−PMA) . Each saTo evaluate the efficacy of the PMA-RT-qPCR assay to selectively detect the infectious CGMMV virus in the presence of inactive CGMMV virus, PMA treatments were performed in predefined ratios of CGMMV virus suspensions . Each sample, containing different numbers of infectious and inactive viruses, was divided into two aliquots and assessed in two different ways: (i) PMA pretreatment followed by RT-qPCR  and (ii) RT-qPCR (three per replicate), respectively. Three replicates were performed for each of the two different assays.A total of 13 leaf, 3 fruit and 6 seedlot samples were collected from CGMMV-infected fields during 2020–2021 . The leaAll naturally infected watermelon leaf, fruit and seedlot samples as well as controls were assessed for the quantities of total or infectious CGMMV virus by RT-qPCR and PMA-RT-qPCR, respectively. In addition, the infection rates of seedlot samples were detected by bioassays.−1 PMA pretreatment followed by RT-qPCR, respectively. Each sample was replicated three times, and each replicate was analyzed by three parallel RT-qPCRs . The average Ct value for each sample was calculated based on the analysis of nine RT-qPCR amplifications.For RT-qPCR and PMA-RT-qPCR, the protocol for preparing CGMMV virus suspensions from infected contaminated leaf, fruit and seedlot samples was based on the methods of Jones et al. (1986) and Zhang et al. (2016) as described previously ,46. The For bioassays, 60 seeds were selected randomly from each of 6 seedlots and sown in 20 cm diameter plastic pots filled with sterile substrate. The pots were kept in a greenhouse at 18–20 °C (night)/26–30 °C (day) with 80–100% relative humidity under natural daylight conditions. Four weeks later, symptoms of leaf mottling and mosaic were checked, and the disease incidence (DI) was examined according to the following equation: DI = no. of diseased plants/total no. of plants × 100%. The bioassay was repeated three times.p < 0.05, was carried out to determine significant differences between the means. Ct and DI values are expressed as the means ± SD.All statistical analyses were performed using SPSS 17.0 software . A one-way analysis of variance (ANOVA), with a significance level of Specificity analyses using the primer pair cp3-1F/cp3-1R showed positive results for all eight isolates of CGMMV virus, while no amplification products were obtained from the other related virus isolates of WMV, CMV, MMV, TRSV, CCYV, ZYMV, SqMV, MYSV, MNSV, PNRSV, PRSV, TMV and PVY . The amp8 to 1.57 × 10 copies·μL−1. A good linear relationship between the log of the concentration of CGMMV plasmid and the corresponding Ct values was obtained  (2 copies·μL−1 per reaction produced good reproducibility (CV < 35), and the corresponding Ct value was 31.33. A lower concentration of CGMMV plasmid 1.57 × 10 copies·μL−1 per reaction resulted in poor reproducibility (CV > 35), giving a Ct value of 35.43. Hence, the detection limit of the RT-qPCR assay was 1.57 × 102 copies·μL−1 of CGMMV plasmid.A quantification standard curve was constructed by a dilution series of CGMMV plasmid ranging from 1.57 × 10 0.9987) . The ampIn the PMA-RT-qPCR assay, an adequate concentration of PMA should be added to inhibit the amplification of target RNA from inactive CGMMV virus and quantify target RNA from infectious CGMMV virus. Hence, the efficiency and toxicity of a range of PMA concentrations were tested with infectious and inactive virus suspensions of CGMMV.−1, the Ct values increased significantly (p < 0.05), which suggested that the inhibition of amplification of the RNA from inactive virus significantly increased with increasing PMA concentration. When the PMA concentration was equal to or greater than 120 μmol·L−1, the Ct values were greater than 31 and remained constant, implying that the application of RNA from inactive CGMMV was completely prevented , which suggested that PMA had a similar performance at these concentrations. However, when the PMA concentration increased from 120 to 200 μmol·L−1, the Ct values increased significantly (p < 0.05), indicating that high PMA concentrations (> 120 μmol·L−1) exerted toxicity toward the infectious CGMMV virus . For PMA-RT-qPCR, as the ratio of infectious virus increased from 0% to 100%, the Ct values decreased gradually from 34.38 ± 0.81 to 16.54 ± 0.45 (p < 0.05)  were assessed by RT-qPCR and PMA-RT-qPCR assays. For RT-qPCR, when the ratios of infectious virus increased from 0% to 100%, the Ct values remained nearly unchanged in the range of 16.31 ± 0.42 to 17.60 ± 0.80 ( < 0.05) A. The dC < 0.05) B. dCt va2~1.54 × 106 copies per gram. The Ct value detected by PMA-RT-qPCR was higher than that detected by RT-qPCR, which was in the range of 20.48~31.37, and the corresponding concentration of active CGMMV virus was 2.08 × 102~5.08 × 105 copies per gram , Yingkou City, Liaoning Province (sample 5) and Zhongwei City, Ningxia Hui Autonomous Region (Samples 8 and 11) gave Ct values ranging from 23.27 ± 0.83 to 28.01 ± 1.40, corresponding to CGMMV copy numbers of 2.30 × 103 to 6.89 × 104 copies per gram. The quantities of infectious CGMMV virus were fairly low in leaf and fruit samples originating from Ningbo City, Zhejiang Province (samples 3 and 10) and Sanya City, Hainan Province (sample 7), with 5.24 × 102 to 7.04 × 102 copies per gram  and Sanya City, Hainan Province (sample 6), which yielded Ct values of 21.37 ± 0.82 and 20.48 ± 0.47 by PMA-RT-qPCR, equivalent to 2.68 × 10per gram .3 and 2.08 × 102 copies per gram of seeds, respectively , which gave Ct values of 28.50 ± 0.46 and 31.37 ± 0.42 by PMA-RT-qPCR, carrying 1.63 × 10ectively . In the ectively , with DIIn recent years, CGMMV has emerged as a devastating virus of cucurbit crops that can cause substantial economic losses worldwide ,17,48. TSeveral molecular methods, e.g., RT-PCR, IC-RT-PCR, RT-LAMP and RT-qPCR, have been developed for the detection of CGMMV ,26,27,28cp gene, which produced strong positive results for all eight CGMMV isolates, while no signals were obtained from the other 13 virus isolates tested. In the RT-qPCR assay, when the concentration of CGMMV plasmid DNA was less than 1.57 × 102 copies·μL−1, the Ct values were greater than 31, which indicated that the detection limit of the RT-qPCR assay was 102 copies·μL−1 of CGMMV plasmid.In the present study, the RT-qPCR specific primers cp3-1F and cp3-1R were developed for the specific detection of CGMMV based on the −1. PMA-RT-qPCR has been applied to detect active human and environmental viruses in several previous studies [−1. For the detection of infectious SARS-CoV-2, the optimal PMA concentration was 50 μmol·L−1 coupled with 0.005% SDS [−1 [The PMA concentration is the main key factor that affects the efficiency of the PMA-RT-qPCR assay to discriminate between infectious and inactive viruses. In this study, the concentration of PMA was optimized to distinguish between infectious and inactive CGMMV viruses. The optimal PMA concentration was 120 μmol·L studies ,39,40,50 SDS [−1 . Althoug SDS [−1 ,39,41,50The consistency and reliability of the PMA-RT-qPCR assay were confirmed. When the virus samples were detected directly by RT-qPCR, no significant differences were found among the different defined ratios of virus suspensions . However, after PMA pretreatment, the Ct values decreased significantly as the proportion of infectious CGMMV virus increased from 0% to 100%. Correspondingly, the dCt values also increased gradually. However, due to the difficulty of plant virus culture, virus suspensions prepared from infected tissues may contain both infectious and noninfectious virus; therefore, the maximum value of dCt could not be detected based on fully viable and completely inactive CGMMV virus samples. To minimize the unwanted interference, infected tissues were freshly collected, and virus suspensions were prepared immediately. The method established in this study was feasible for the mixed detection of infectious and inactive virus samples with a defined ratio. In addition, it has been shown that surfactants can optimize RT-qPCR results by weakening the viral protein envelope ,52,53, mThe PMA-RT-qPCR assay was further employed for the selective detection of infectious CGMMV viruses in naturally infected watermelon leaves, fruits and seedlots. In total, 22 watermelon tissue samples suspected to be infected with CGMMV were collected from different parts of China and detected by PMA-RT-qPCR. Quantifiable CGMMV viruses were found in eight leaf samples, three fruit samples and two seedlot samples, indicating that infectious CGMMV viruses could be detected from naturally infected leaves, fruits and seeds.In conclusion, the PMA-RT-qPCR assay developed in the current study was an effective, sensitive and reliable assay to quantitatively detect infectious CGMMV virus from leaves, fruits and seedlots of cucurbit crops. Therefore, the PMA-RT-qPCR assay can serve as a useful tool to be used for monitoring and early warning of CGMMV virus for commercial cucurbit producers.


Aedes aegypti is the most dominant vector in the transmission of dengue hemorrhagic fever (DHF). In addition to Ae. aegypti, Ae. albopictus is a secondary vector of the dengue virus, and both species are widespread in Indonesia. The dengue virus is transmitted from person to person through the bite of an Aedes spp. The vertical  transmission of the dengue virus from infective female mosquitoes to their offspring is one of the means by which the dengue virus maintains its existence in nature. Transovarial dengue virus transmission in Aedes spp. mosquitoes contributes to the spread and maintenance of the dengue epidemic. This study employed a qualitative survey to detect dengue virus transovarial transmission in Ternate using the streptavidin-biotin-peroxidase complex (ISBPC) immunohistochemical test. The ISBPC examination of samples collected from the four subdistricts in Ternate revealed a positive result for transovarial transmission of dengue virus. Four Aedes spp., including two Ae. aegypti females, one Ae. albopictus female, and one Ae. albopictus male, tested positive for transovarial transmission of dengue virus in the district of North Ternate. Four Aedes spp., including three Ae. aegypti females and one Ae. aegypti male, were found to be positive for the transovarial transmission of dengue virus in the Central Ternate district. Seven Aedes spp., including five Ae. aegypti females, one Ae. aegypti male, and one Ae. albopictus female, tested positive for transovarial transmission of the dengue virus in the district of South Ternate city. One Ae. aegypti male showed positive results for transovarial transmission of dengue virus in the Ternate Island District. In this study, the transovarial transmission of the dengue virus occurred in both Aedes spp. female and male mosquitoes. It was demonstrated that Aedes spp. carry the dengue virus in their ovaries and can pass it on to their offspring. As a result, the cycle of passing the dengue virus on to local mosquito populations in the city of Ternate is not going to end just yet. Aedes aegypti is by far the most significant agent in the dissemination of DHF, whereas Ae. albopictus is the second, less effective, vector [Dengue hemorrhagic fever (DHF) is an arbovirus disease caused by the dengue virus, which belongs to the Flaviviridae family . Aedes a, vector . The mos, vector .The mosquitoes are able to continue their life cycle despite exposure to the dengue virus because the virus does not cause any cytopathic effect . Two mecAe. aegypti contributes to the spread and maintenance of the dengue epidemic [Initially, it was believed that transovarial transmission of the dengue virus did not play a role in the epidemiology of dengue transmission. Recent evidence suggests that transovarial transmission of the dengue virus in epidemic . Immunocepidemic . Dengue epidemic ,9. Aedes spp. can serve as an alternative early warning system in vector control for predicting human dengue virus transmission. Since there are no data available regarding the transovarial transmission of the dengue virus in this city, it is imperative that research be conducted into the mechanism. An investigation into the possibility of transovarial transmission was carried out using the streptavidin-biotin-peroxidase complex (ISBPC) immunohistochemical test. It is hoped that up-to-date information can be uncovered through this study, which can be used as a guide for bolstering effective and sustainable vector control management in lowering dengue fever-related morbidity and mortality.Ternate, in North Maluku province, is one of the cities in Indonesia where dengue fever is currently endemic. Cases of DHF occur nearly every year, particularly in the city of Ternate. Detection of transovarial transmission of dengue virus in This study was conducted in Ternate City, which is located in the province of North Maluku, Indonesia. The larvae used in this study were in the third or fourth instar. Larvae were collected from four subdistricts in an endemic village of DHF in the city of Ternate. The larvae were bred until imago stage. The collected larvae were placed on a tray. The larvae were given 20 mg of dog food per day and this was increased according to the number and size of the larvae. Every day, dead larvae were removed using a pipette. Individual larvae were scooped or captured with a pipette and transferred to another tray to replace the dirty water in the original tray. When the larvae matured into pupae, they were placed in a plastic tray filled with water before being transferred to the mosquito cage for rearing. Therefore, if the pupa matured into an adult mosquito, it was already contained in the mosquito cage. The dead mosquitoes in the cages were removed daily with an aspirator and the cages were cleaned weekly with a wet cloth. Seven-day-old imagoes were collected using an aspirator and put into a jar for storage.The ISBPC immunohistochemical assay was utilized to examine mosquito head press preparations for the presence of the dengue virus. The examination was carried out at the Parasitology Laboratory of the Faculty of Medicine, Sam Ratulangi University. The head of the adult mosquito was separated from the body and then pressed on the object glass. Cold methanol was used to fix the preparations on the object glass at 20 °C for 3–5 min. The preparations were briefly washed under running water before being treated with a peroxidase blocking solution to eliminate endogenous peroxidase activity for 10 min at room temperature. The prepared commercial monoclonal antibody  pH 7.4) was added at a maximum of 100 µL per preparation  and incubated for 24 h in the refrigerator. The preparations were then washed for 5 min with PBS. After adding up to 100 µL of biotinylated universal secondary antibody per preparation, they were incubated at room temperature (25 °C) for 10 min. The preparations were washed with PBS for an additional five minutes. They were then incubated for 10 min with a ready-to-use streptavidin/peroxidase complex reagent. Following that, the preparations were washed with PBS for 5 min. The preparations were examined using a microscope with a 400× magnification. The head of an uninfected mosquito was used as a control.Ae. aegypti and Ae. albopictus with a commercial anti-dengue monoclonal antibody revealed immunoreaction of the preparation  the infectious female mosquito feeds on the blood of the host by biting them and sucking their blood. This ripens the eggs and allows the virus to reproduce (replicate) in the mid-gut of the larvae, which makes them infectious ; (2) nonAe. aegypti that survived to the seventh generation, transovarial dengue virus infection was still detected (12.6%); infection in female mosquitoes (14.8%) was higher than in male mosquitoes (10%) [The dengue virus will propagate in the embryos inside the eggs of an infective-gravid female mosquito. Infected eggs could possibly endure for months . After hes (10%) .Ae. albopictus was detected 7–41 days prior to the first reported clinical case of DHF in human [Aedes spp. persists, the chain of dengue virus transmission in mosquitoes will continue. Transovarial dengue virus infection in Ae. aegypti and Ae. albopictus can be found throughout the year regardless of the season. It is obvious that transovarial transmission can be viewed as a reservoir for virus survival in nature.It was initially believed that transovarial transmission of the dengue virus played no role in the epidemiology of dengue. One study indicates that transovarial transmission of the dengue virus in in human . If the Aedes spp. mosquitoes. Although male mosquitoes do not feed on the blood of infected humans, they can still become infected with the dengue virus through transmission from infected female parent. It is possible for a dengue virus infection to be passed on to the next generation if an infected male mosquito mates with a dengue virus-negative female mosquito. This will result in the generation of dengue virus-positive offspring. It was demonstrated that Aedes spp. carry the dengue virus in their ovaries and can pass it on to their offspring. If the Aedes spp. mosquito population is not completely eradicated, the cycle of transmission of the dengue virus from humans to mosquitoes in the city of Ternate will continue unabated.In this study, the transovarial transmission of the dengue virus occurred in both male and female Ae. aegypti and Ae. albopictus, both of which are known to transmit dengue fever in Ternate, Indonesia. The district of South Ternate exhibited the highest levels of transovarial transmission. In Ternate City, transovarial transmission was discovered not only in female but also in male Aedes mosquitoes. The existence of transovarial transmission of dengue virus by Aedes spp. mosquitoes suggests that the mosquito-borne transmission of dengue virus in Ternate City is continuing. This transovarial transmission contributes to the spread and maintenance of the dengue virus in nature. This is because mosquitoes that are infected through transovarial transmission have the ability to transmit the dengue virus, which is present in their salivary glands, when they bite healthy humans. Therefore, it is recommended that the management of mosquito vector eradication in endemic areas should be carried out comprehensively by taking into account the potential for transovarial transmission of the dengue virus.This study provided conclusive evidence that the transovarial transmission of the dengue virus occurs in


In the last two decades, the (re)emergence of zoonotic viruses  has resulted in devastating consequences from a health, economic, and social perspective. Changes in ecological and environmental factors, demographics, and socio-economic behavior have increased the risk of spillover events and of (re)emergence of zoonotic viruses  that mediate critical steps in virus replication and immune evasion. Thus, PPIs are prime targets for the development of therapeutics and vaccines. However, their characterization is an urgent albeit challenging task that benefits drastically from the integration of computational and experimental approaches. This Research Topic brings together nine articles  that collectively leverage experimentally and computationally derived information to describe important biological processes mediated by virus-host PPIs.Chung and Song summarize interactions of proteins expressed by oncogenic gammaherpesviruses  with host poly (ADP-Ribose) polymerase 1 (PARP1), a nuclear enzyme that regulates diverse cellular pathways. PARP1's interaction with several viral proteins supports establishment of viral latency by down-regulating viral DNA replication, and reducing virus production to prevent reactivation. Simultaneously, these viruses also employ multiple mechanisms to down-regulate PARP1 expression to then promote their own replication. Fishburn et al. systematically review virus-host PPIs that mediate virus entry and replication of various flaviviruses, including dengue virus (DENV), Zika virus (ZIKV), West Nile virus (WNV), yellow fever virus (YFV), and Japanese encephalitis virus (JEV). The authors also summarize the role of virus-host and intra-host PPIs mediated by cellular proteins involved in autophagy, mitochondrial, and innate immune responses including the antagonism of host immunity. Experimentally determined virus-host PPIs are compiled in PPI databases. Saha et al. give a detailed summary of virus-host PPI repositories and illustrate how publicly available data can be leveraged to identify shared and unique strategies employed by four emerging viruses to co-opt cellular processes. Notably, Saha et al. and Gabriel Valiente emphasize the poor overlap between the different repositories, highlighting the need to use meta-databases that combine different primary resources and inspect annotated PPIs during dataset assembly.Opening this Research Topic, four review articles present an overview of virus-host PPIs and their biological role by focusing on specific host proteins, virus family or providing a more holistic view. Yang et al. summarize technical details of DL in the context of viral-host PPI prediction, including the different types of architecture, dataset preparation, feature engineering and performance assessment. While we expect DL-based methods to play a major role in inter-species PPI prediction in the near future, Yang et al. highlight important aspects of the technique that require further improvements and careful examination.Experimental characterization of the virus-host protein interactome is far from complete and only few viruses have been extensively studied. In order to narrow this knowledge gap, computational tools can provide a catalog of high-confidence PPI predictions to be tested experimentally  protein of the Omicron variant of SARS-CoV-2 and four neutralizing monoclonal antibodies (mAbs) targeting S with known structure. This study highlights amino acid variations that are predicted to decrease mAb-binding affinities without completely abrogating interactions and has important implications in the rapid assessment of neutralization escape potential of emerging viral strains. Zhu et al. experimentally studied the interaction between a PDZ-domain binding motif (PBM) found in SARS-CoV-2 envelope (E) protein and PDZ-containing cellular proteins, which are commonly targeted by other viruses  RNA-dependent RNA polymerase NS5B modulates this viral protein via phosphorylation of conserved residues. Site-directed mutagenesis of key NS5B residues to mimic phosphorylation significantly reduced RNA polymerase activity and prevented rescue of HCV from infectious clones, thus, describing a mechanism of viral polymerase inactivation whose biological role remains to be determined.The following research articles illustrate important aspects of virus-host PPIs, including amino acid variations at protein interfaces, through a wide range of approaches such as X-ray crystallography, cryo-electron microscopy (Cryo-EM), molecular dynamics (MD), protein structure modeling, and binding affinity assays. In conclusion, this Research Topic provides an overview of computational and experimental approaches that, when combined, can significantly accelerate our understanding of virus-host PPIs and their biological role in viral infectious diseases. We are grateful for the valuable contributions of authors, reviewers, and members of the Editorial team at Frontiers.All authors listed have made a substantial, direct, and intellectual contribution to the work and approved it for publication.RKJ would like to acknowledge funding from Louisiana State University Health Sciences Center-Shreveport and National Institute of Allergy and Infectious Diseases of the National Institutes of Health (R21 AI156482).The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Reoviridae. ARV could replicate in its host Locusta migratoria and result in host death. Lower-dose ARV infection affected ovary development and resulted in a significant reduction in fecundity. The identification and characterization of a novel pathogenic reovirus could potentially promote the development of new biological control agents.Grasshoppers can swarm in the millions and destroy crops over wide areas, posing a major economic threat to agriculture. A wide range of insect-related viruses has recently been reported in the metagenomics of grasshoppers. Here, we identified and isolated a novel reovirus from grasshoppers, named Acrididae reovirus (ARV). The complete genome of ARV was composed of nine dsRNA segments. Phylogenetic analysis revealed that ARV formed a monophyletic lineage with unclassified insect-associated reoviruses and was sufficiently distinct from known genera of  Metarhizium acridum and Paranosema locustae have been used in the concerted controlling of grasshoppers in China and have successfully prevented migratory locusts from reaching plague proportions . On. On10]. The virus pellet was transferred to TRIzol  for total viral RNA extraction according to the manufacturer’s instructions. Reverse transcription was performed with the PrimeScript™RT reagent Kit  using random hexamers, and PCR amplicons were carried out using the PrimeSTAR Max DNA Polymerase . To confirm the presence of reovirus, specific primers targeting the RNA-dependent RNA polymerase (RdRp) were designed based on the viral contigs in our published study .Twenty microliters of viral particles were dispensed on carbon-coated copper grids for 5 min, washed, and negatively stained with 3% phosphotungstic acid . Virions were imaged by a Hitachi H-7500 transmission electron microscope .Viral RNA was extracted from purified virions isolated from S2 cell cultures as described above. The RNA was denatured at 95 °C for 3 min to unwind the double strands. Residual Ribosomal RNA (rRNA) was depleted using Ribo-Zero™ kits  before library construction. Libraries were constructed using a TruSeq total RNA library preparation kit  and paired-end (150 bp) sequencing was performed on the Illumina Novaseq 6000 platform (Illumina). Trimming and assembly of sequencing reads, virus discovery, and calculation of virus abundance were carried out as described in a previous study . Reoviru50 assay failed to titrate the viral particles because ARV did not induce a cytopathic effect in insect cell cultures. The Quantitative Real-Time PCR (qRT-PCR) methodology had been proven effective in the determination of reovirus stock titers [A classical TCIDk titers ,20. The k titers ,20. BrieLocust migratoria was a natural host of reovirus and was easy to raise in the laboratory. Newly molted 3rd-instar L. migratoria nymphs were therefore injected with 9 × 1012 virions into the membrane between the third and fourth abdominal segments using a Nanoinjector . An equal volume of sterile PBS was injected as the control. The survival of inoculated locusts was recorded daily until no deaths occurred. The qPCR method was used to demonstrate the successful infection of the reovirus in locusts. Six replicates with twenty-five nymphs for each inoculation were generated.10 virions to increase nymph survival for other pathogenic effects characterization. Guts, ovaries, and heads of infected samples were dissected at 9 time points across all developmental stages, including 1, 3, 5, 7, 9, 13, 17, 21, 24, and 27 days post-infection. RT-PCR was carried out on RNA extracted from guts, ovaries, and heads of infected and uninfected locusts. qPCR was used to test for viral titers. The viral load of a mixture of fifteen individuals was quantified at each time point. Experiments were repeated five times. The infection of the ovaries by the reovirus may affect the reproduction and vertical transmission of locusts, so ovary sizes were measured and compared as an indication of reproduction ability. Moreover, the egg-laying amount of fifteen pairs of locusts was recorded, and the experiments were repeated four times.Furthermore, 3rd-instar locust nymphs were then injected with a lower dose at 9 × 10To reveal the transmission mode of ARV, twenty-five 3rd-instar nymphs were exposed to the feces of virus-infected locusts for 2 h, and the viral load of the locusts was measured after 24 h. Additionally, eggs laid by infected adults were collected. Total RNA was extracted from two types of eggs, including those that had not been cleaned and had been disinfected with sodium hypochlorite. Viral loads were also measured for nymphs hatched from cleaned or uncleaned eggs. qPCR was used to test for viral titers of eggs. Experiments were repeated 5 times.p < 0.05; specific p-values were provided in the text. Statistical analysis was performed using IBM SPSS Statistics 26.0  and GraphPad Prism 8.0.1 .To compare the qualitative and quantitative variables between groups, Student’s T-test or the Mann–Whitney U test was used in a univariate analysis. The results were considered statistically significant at https://talk.ictvonline.org/ictv-reports, accessed on 1 June 2022). Considering the host origin of the virus, this novel reovirus was named Acrididae reovirus (ARV).RdRp sequences of the reovirus can be successfully amplified from field-collected samples from these four species. Reovirus RdRp of grasshoppers only shared a 30% amino acid (aa) identity with its most closely related RdRp sequence of Hubei reo-like virus 1. A reovirus of grasshoppers can be considered a novel insect-specific virus based on ICTV species demarcation criteria  of 94% and a correlation coefficient (R2) of 1. The viral genomic RNA level in purified virions and various tissues of infected nymphs were further quantified through the extrapolation of Ct-values to the standard curve in the real-time PCR analysis.The Quantitative Real-Time PCR method was used to quantify the genomic RNA level of purified ARV virions . The tra12 virions) ARV has a severe pathogenic effect on L. migratoria. ARV-infected 3rd instar nymphs started to die at 3 days post-infection (dpi). The death rate was highest within 3–8 dpi, and viral loads reached a peak (5.99 × 1018 virions/μL). The final survival rate for the infected locusts was 19%  of ARV, which would not cause early mortality, was injected into 3rd instar locusts to characterize virus replication and pathology in different tissues. ARV was detected in the guts, heads, and ovaries of infected locusts  have been studied for their potential as biopesticides due to their induction of sublethal effects in infected lepidopterans, including lower weight and shorter adult lifespan [Diadromus pulchellus idnoreovirus 1 can interact with ascovirus to down-regulate host immunity for successful parasitism by inhibiting the melanization of the D. pulchellus [L. migratoria. ARV can be successfully cultured in insect cell cultures and live locusts, which makes it a good candidate to be developed as a biocontrol agent. This is of particular interest for the increasing demand for environmentally friendly novel agents to manage the frequent outbreaks of locust plagues worldwide.The current known hosts of insect-specific reoviruses are almost exclusively limited to lepidoptera, hymenoptera, and diptera insects . Cytoplalifespan ,23. Diadlchellus ,24. To dReoviridae. According to current ICTV criteria, the identities of the RdRp amino acid sequence between different genera are generally less than 30%. Therefore, ARV with other unclassified insect reoviruses appears to be a novel genus of Reoviridae. Furthermore, they form a distinct clade that closely groups with plant-infecting fijiviruses. This discovery will contribute to deciphering the evolution of Reoviridae.ARV contains only nine dsRNA segments. Additionally, we reveal a conserved hexanucleotide terminus at the 3′ ends that is different from other reoviruses. The conserved terminal sequences of reoviruses play important roles in RNA replication, stability, and immune recognition ,26. PhylDiaphorina citri reovirus (DcRV) was recently identified from Diaphorina citri populations and considered a novel species of Reoviridae [Bombyx mori cytoplasmic polyhedrosis virus (BmCPV) is a relatively well-studied reovirus. A recent study discovers that ROS induced by vSP27 can suppress BmCPV infection via the NF-κB signaling pathway. vSP27 is translated from a BmCPV-derived circular RNA (circRNA-vSP27) [The study of virus pathogenicity and its interaction with hosts is often limited by the difficulty of virus isolation and culture. For example, oviridae . Notablyoviridae . Bombyx A-vSP27) . FortunaL. migratoria. A lower dose of ARV can jeopardize ovarian development, thus reducing the reproduction of infected hosts. Both features are useful in controlling locust populations in the field. More importantly, given it is found in multiple grasshopper species, it may have wider usage as a biocontrol agent to manage orthopteran pests.To determine the optimal proposal to apply virus agents in the field, it is important to acquire the most knowledge concerning virus replication dynamics in the hosts. We show that high-dose ARV significantly reduced the survival of infected One aspect of ARV replication dynamics that deserves attention is the maintenance of the virus in the host population, indicating persistent infection. Persistent infection is a consequence of interactions between the viral modulation of replication and trade-offs of host immunity ,31. TherIn this work, we identified and isolated a novel reovirus from grasshoppers, named Acrididae reovirus (ARV). ARV can be successfully cultured in insect cell cultures and live locusts. Using genome sequencing, our results showed that the complete genome of ARV comprised nine segments. All genomic segments of ARV shared a conserved hexanucleotide terminus at their 3′ ends (CGCGAC-3′), which was different from previously reported reoviruses. Importantly, ARV was pathogenic to its host by reducing host survival and reproduction. These results indicated that ARV can be a good candidate for the biological control of grasshopper pests.


To review and analyse evidence regarding costs for retinopathy of prematurity (ROP) screening, lifetime costs and resource use among infants born preterm who develop ROP, and how these costs have developed over time in different regions.Systematic review and meta-analysisPubMed and Scopus from inception to 23 June 2021.Included studies presented costs for ROP screening and the lifetime costs (including laser treatment and follow-up costs) and resource use among people who develop ROP. Studies not reporting on cost calculation methods or ROP-specific costs were excluded.Two independent reviewers screened for inclusion and extracted data, including items from a published checklist for quality assessment used for bias assessment, summary and random-effects meta-analysis for treatment costs. Included studies were further searched to identify eligible references and citations.In total, 15 studies reported ROP screening costs, and 13 reported lifetime costs (either treatment and/or follow-up costs) for infants with ROP. The range for screening costs (10 studies) was US$5–US$253 per visit, or US$324–US$1072 per screened child (5 studies). Costs for treatment (11 studies) ranged from US$38 to US$6500 per child. Four studies reported healthcare follow-up costs , and of these, three also reported lifetime costs for blindness (range US$26 686–US$224 295) using secondary cost data. Included papers largely followed the quality assessment checklist items, thus indicating a low risk of bias.The costs of screening for and treating ROP are small compared with the societal costs of resulting blindness. However, little evidence is available for predicting the effects of changes in patient population, screening schedule or ROP treatments.CRD42020208213. PubMed and Scopus were searched systematically.Since manual search of reference lists and citations of the identified papers did not identify additional studies, the database search had good coverage of the topic of investigation.The main limitations of this work were the exclusion of grey literature and the lack of analyses of publication bias for the meta-analysis.Where lack of variance information in included studies hindered meta-analysis, guidance for synthesis in systematic reviews without meta-analyses were followed.Improvements in neonatal care have resulted in increased survival among children born preterm,2ROP management and treatment economics are still challenging in many health systems because of screening-associated costs, patient-related costs and medicolegal liability.6Here, we present an overview of costs associated with ROP screening and treatment, examining the evidence related to costs for ROP screening and lifetime costs (including laser treatment and follow-up costs) and resource use among infants born preterm who develop ROP. We also examine the trajectories of these costs over time in different regions in a meta-analysis.This work followed the Preferred Reporting Items for Systematic Review and Meta-Analysis ,8PubMed and Scopus were searched (10.1136/bmjopen-2021-057864.supp1Supplementary dataRayyan QCRI was used for handling duplicates and the selection of studies for inclusion. Two independent reviewers (JH and CL or HG) searched the databases, screened articles for eligibility, extracted data using a prespecified data extraction sheet , and hanThe data extraction sheet included items  from a p10Conventional screening (excluding telemedicine costs), laser treatment and long-term follow-up costs were reported, respectively, accounting for ROP severity and differences over time and between countries. Identified costs were adjusted to 2020 US dollars (US$) using annual exchange ratesThis project did not include patient or public involvement in developing the research questions, design, conduct, choice of outcome measures or recruitment.Of the 503 studies screened after duplicates from the databases were removed, 123 were assessed for eligibility based on full text, and 19 studies were included in the synthesis of results . ReasonsTwelve studies were conducted in high-income economies: seven in the USA, two in Canada and one each in the UK, Netherlands and France. Three studies were conducted in upper-middle-income economies: one each in Peru, Thailand and Brazil. Three studies were conducted in lower-middle-income economies: two in India and one in Iran. One study was conducted in both the United States and Mexico . All stuThe quality assessment indicated a high overall quality of the included studies , with 16Studies reporting costs related to screening had different designs: six were retrospective cohort studies using medical chart review or register data,et al25 28 29Costs for routine ROP screening, excluding transportation costs, are reported in et alet alJavitt et alet alet alIn studies comparing alternative screening or treatment options, no common comparator was identified. The incremental cost reported in Black In all, 14 studies reported costs related to the laser treatment of ROP . Four stet alet al.et alEleven studies reported total treatment costs per child, at a mean US$2442 (range: US$38–US$6500). Castillo-Riquelme 2 and Cochrane Q) indicated high heterogeneity between studies within each country classification, which suggests that the results from the meta-analysis should be interpreted with caution.Accounting for the low assessed risk of bias but large expected variation based on cost-levels of individual countries, the meta-analysis by country classification  estimateet alet alet alet alet alOnly four studies reported follow-up costs occurring after screening and treatment, and although the risk of bias was assessed as low, the reported results largely differed between studies. Castillo-Riquelme The studies we identified could be grouped by whether they reported costs for screening, costs for treatment or costs (and QALYs) during long-term follow-up or even from a lifetime perspective. The cost range per ROP screening was US$5–US$253 per visit, or US$324–US$1072 per screened child. Costs for ROP treatment ranged from US$38–US$6500 per child. In addition, four studies reported healthcare follow-up costs, and three reported lifetime costs using secondary data on costs for blindness. Although quality assessment indicated a low risk of bias, comparisons between studies were challenging because of the lack of detailed cost and resource use data.To our knowledge, this is the first systematic review of ROP costs. Included papers largely followed the quality assessment checklist items of a commonly used tool,Cost components for ROP screening included staff salaries/time, equipment and maintenance, supplies and staff training. Screening costs for ROP were low compared with other associated costs and, with few exceptions, of the same order of magnitude in the included studies. Exceptions were probably attributable to salary differences.et al,Screening access and schedules vary between countries.et alet alTreatment costs were low compared with the costs for follow-up, with Brazil, Mexico and Peru having substantially lower treatment costs than the other countries. Both Javitt Variation among studies in whether one or two eyes were treated made comparisons less relevant, which may reflect the unilateral schedule used in the historically influential Cryo-ROP study.47When examining ROP treatment, cost components included staff salaries/time, equipment and maintenance, supplies and staff training. Sometimes anaesthesia costs were reported separately or excluded. Transportation was also a considerable cost component in relation to treatment.et alet alet al included transportation costs, white canes, Braille equipment and supplies,Although ROP results in high costs throughout life, this outcome is primarily based on secondary data for blindness. As the leading cause of preventable childhood blindnessThere is a need for comprehensive knowledge about the costs of ROP, both during the introduction of new ROP screening programmes and in countries with established programmes that are now redistributing resources to handle the increasing survival of very preterm infants with high disease burden. In addition to relevant cost components of ROP , complemAlthough costs of screening and treating ROP are substantial for health systems, they are small compared with the follow-up costs to society of resulting blindness. However, little evidence is available to support predictions about the consequences of changes in the patient population, screening schedule or treatment regimens for ROP.


The Eastern grey squirrel represents a huge threat to the conservation of the native Eurasian red squirrel when the two species coexist. Moreover, given their confident behaviour with humans, the non-native squirrels can negatively impact public health. The U-SAVEREDS Project was set up for Eurasian red squirrel conservation in Umbria through the eradication of the alien species and it also provided information on the health status of the Eastern grey squirrel to identify any infectious agents. The recovery of zoonotic pathogens allowed to assess the Eastern grey squirrel’s impact on human and domestic and wild animals’ health, provide helpful feedback for the management and eradication procedures, and raise public awareness through environmental education.Invasive alien species are non-native species introduced deliberately or unintentionally beyond their past or present natural distribution, and their introduction and spread threatens local biological diversity. The Eastern grey squirrel is native to North America and was introduced to the British Islands, Italy, and South Africa. Around the year 2000, a new population of grey squirrels was recorded in Perugia, central Italy, where the species populated an area of approximately 50 kmSciurus carolinensis) in Europe is one of the best-known cases of invasive alien species (IAS) colonisation, that poses a severe risk to the conservation of biodiversity. In 2003, it was released in a private wildlife park near the city of Perugia , where it is replacing the native Eurasian red squirrel (Sciurus vulgaris). The LIFE13 BIO/IT/000204 Project (U-SAVEREDS) was set up for the Sciurus vulgaris conservation in Umbria through an eradication campaign of grey squirrels. One hundred and fifty-four animals were analysed for bacteriological, mycological, virological, and serological investigations (C4 action). Sanitary screening showed that Sciurus carolinensis is a dermatophyte carrier, and therefore, it could cause public health issues for humans, considering its confident behaviour. Moreover, it has been marginally responsible for the spreading of Candida albicans, Coxiella burnetii, and Borrelia lusitaniae. Health status evaluation conducted on the Sciurus carolinensis population indicated that it is necessary to raise awareness of its impacts on biodiversity and human health. Moreover, the health status and behaviours of the IAS must be considered when control or eradication campaigns are planned.The introduction of the Eastern grey squirrel ( Invasive alien species (IAS) are non-native species introduced deliberately or unintentionally beyond their past or present natural distribution, and their introduction and spread threatens local biological diversity . The gloSciurus carolinensis) [2 and is characterised by mixed deciduous forests and urban centres. This alien species is replacing the native Eurasian red squirrel (Sciurus vulgaris) in the British Islands and Italy through a complex process known as ‘exploitation competition’ [Chlamydia sp. [Toxoplasma gondii [Leptospira sp. [Francisella tularensis [Borrelia sp., Babesia sp., and Rickettsia sp. [One of the best-known cases of invasive species colonisation in Europe is the introduction of the Eastern grey squirrel (inensis) ,13,14. Tinensis) . In Umbrinensis) . Its spretition’ . In the etition’ ,19. The etition’ . By contetition’ ,22. Theretition’ . The SQPetition’ , Englandetition’ , Wales, etition’ , but theetition’ . Squirreetition’ ,29, yeasetition’ , Chlamydydia sp. , Toxoplaa gondii ,33,34, Lpira sp. ,36, Franlarensis , Borrelitsia sp. ,39,40, atsia sp. , Germanytsia sp. , Northertsia sp. , and Itatsia sp. .The expansion of the grey squirrel population was recognised as a threat to the Eurasian red squirrel by the European Strategy on IAS , and it Sciurus carolinensis, suggesting its relationship with other wildlife species and its role as a carrier of zoonotic agents. The presence of non-native squirrels in popular areas and their greater confident behaviour with humans could have critical implications on public health. Dermatophytoses are one of the most frequent skin diseases of humans and animals. A closely relation between them, especially in urban parks, could favour the onset of the disease by direct or indirect contact. This happens particularly in children and vulnerable subjects of all ages with impaired immune function. To achieve the right form of awareness, it is important to educate communities about zoonoses [The LIFE13 BIO/IT/000204 Project: ‘Management of grey squirrel in Umbria: conservation of red squirrel and preventing loss of biodiversity in Apennines’ , was setzoonoses ,49,50,51zoonoses ).A total of 1070 grey squirrels were removed since October 2014 to September 2018 from the study area of the LIFE U-SAVEREDS project, as described below. Mechanical Tomahawk live traps  baited with walnuts were placed opportunistically or following a regular network, according to the characteristics of each trapping site. They were numbered, geo-referenced, tagged, and placed at the base of tree trunks or on tree branches to avoid disturbance and entry of other wild or domestic species as much as possible. Moreover, they were placed away from sunlight or busy roads to limit additional stress to the captured squirrels and were checked at least twice a day. WIN EPISCOPE, version 2.0 software [A minimum of 271 squirrels was established to be submitted to the health evaluation. The total number was calculated using software , estimatThe management plan was carried out in accordance with the 92/43/CEE Directive, European Union Regulation No. 1143/2014, and the National Hunting Law 157/92 , with positive evaluation by the Italian Institute for Environmental Protection and Research, ISPRA .Sciurus carolinensis population. Each carcass was then placed in a sealed plastic bag and subjected to necropsy at the Istituto Zooprofilattico Sperimentale dell’Umbria e delle Marche ‘Togo Rosati’ (IZSUM). TM VirkonTM  and sterilised under UV light.Euthanasia was performed using carbon dioxide, following the 1099/09/EC Regulation and American Veterinary Medical Association guidelines on animal welfare . This me® ID, bioMérieux, Craponne, France). For anaerobic incubation, samples were also spread on sheep blood agar plates placed in an anaerobic jar with AnaeroGen Compact  and incubated at 37 °C for 18–24 h. Incubation was prolonged for one more day if no colonies appeared within 24 h. Rectal swabs and pulmonary, central nervous system (CNS), and hepatic samples were aseptically collected for standard bacteriological examinations . The samViral isolation was carried out from oral swabs on Vero cell cultures (ATCC CRL-1586 VERO C1008) at the Biosafety Level 3 (BSL-3) laboratory at the Istituto Zooprofilattico Sperimentale Abruzzo e Molise ‘Giuseppe Caporale’ (IZSAM). Vero cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics and 10% foetal calf serum. Monolayer cultures were inoculated with swab material and examined daily for the evidence of cytopathic effect (CPE) for 5–6 days. Three subsequent blind passages were carried out for each sample to allow CPE to appear. ®, Hilden, Germany) following a modified Gram-positive protocol , and subjected to a hemi-nested PCR test [®). PCR-positive products were purified using the QIAquick PCR Purification Kit (QIAGEN®). Sequencing was performed using the BigDye Terminator v1.1 Cycle Sequencing Kit , and reactions were separated through an ABI PRISM 310 Genetic Analyzer . Consensus sequences were created by BioEdit Sequence Alignment Editor, version 7.0.9.0 software and aligned against those from the GenBank database.Mycological examinations were performed to investigate the dermatophytes and yeasts . Hair saPCR test ,58. The ® ID 32 C . Oral, rectal, and vaginal swabs were collected from each animal and plated on Sabouraud dextrose agar, followed by incubation at 37 °C for 24 h. Fungal growth attributable to yeasts was further investigated using API®, Penzberg, Germany) and the QIAamp Viral RNA Mini Kit (QIAGEN®), respectively. As listed in Rickettsia sp. [Babesia sp. [Anaplasma sp. [Coxiella burnetii [Borrelia burgdoferi sensu latu [B. burgdoferi sensu latu were confirmed through end-point PCR tests [®), were subjected to Pox Virus-specific nested PCR tests [Hepatitis E Virus (HEV) [West Nile Virus (WNV) [Genomic DNA and viral RNA were extracted from the spleen and liver samples using the High pure PCR template preparation kit   and Westus (WNV)  detectioToxoplasma gondii, Leptospira interrogans, Francisella tularensis, Chlamydia sp., hantaviruses, and flaviviruses were investigated in the serum of each captured animal  by mixiL. interrogans was performed using the microagglutination test (MAT). Each serum sample was tested against eight locally circulating L. interrogans serovars, namely, icterohaemorrhagiae, canicola, tarassovi, bratislava, grippotiphosa, ballum, hardjo, and pomona. Serum was considered positive at a 1:100 dilution for the tested antigen if at least 50% of Leptospira were agglutinated compared with a control antigen. MAT was also used for the serological diagnosis of F. tularensis, considering positive titres of ≥1:128 [Serological diagnosis of Chlamydia sp. antibodies were investigated by using the complement-fixation test with standardised Serion CFT reagents, according to the manufacturer’s instructions. Sera with 50–100% inhibition of haemolysis at a 1:10 dilution were considered positive.Anti-Puumalavirus (PUUV) and Dobravavirus (DOBV), were performed using the ReaScan Ab-Dect PUUMALA IgG immunochromatography test and the Ab-Dect Dobrava-Hantaan IgG EIA ELISA kit , respectively, following the manufacturer’s instructions. Serological assays for flaviviruses were carried out using the competitive ELISA INgezim WNV Compac , which cross-reacts with different flaviviruses, and the positive samples were further analysed by using serum neutralisation (SN) assays for WNV and Usutu virus (USUV). Serological tests for hantaviruses, particularly the p < 0.05. All analyses were performed using the Stata software .The Fisher’s exact test was used to assess differences in positivity by season, gender, and presence of lesions. The Pearson’s Chi-square test was used to evaluate the differences in positivity for MUs. Statistical significance was set at Out of the 271 foreseen, a total of 154 squirrels  were subjected to necropsy and health investigations. The discrepancy between the hypothesised and the analysed squirrels was because they were not distributed over all 70 MUs, as initially considered. Most trapped animals appeared to be healthy and free from macroscopic internal or external lesions, except eight animals (5.2%) that showed non-exudative alopecic lesions.Nannizzia racemosa , Trichophyton mentagrophytes , Trichophyton thuringiense , and Trichophyton ajelloi . The ITS sequence analysis showed a degree of similarity between 99% and 100%. No significant differences were found between being tested positive for dermatophytes and the presence of lesions (p = 0.257) or gender (p = 0.163). Dermatophyte-positive squirrels were more likely to be found in autumn (p = 0.027) than in other seasons . Moreover, the presence of dermatophyte-positive squirrels was found to be higher in MU 28 than in the other MUs , comprising 30 males and 18 females . ConventBorrelialusitaniae, the causative agent of Lyme’s disease, was detected in a N. racemosa-positive squirrel, originating from MU 26. Coxiella burnetii, the Q fever causative agent, was detected in two N. racemosa-positive squirrels, originating from MU 28. None of the remaining infectious agents were found on molecular analysis.Candida albicans was detected in oral, rectal, and vaginal swabs collected from four animals originating from different MUs , which tested negative for dermatophytes. Moreover, Escherichia coli was isolated from 180 rectal swabs, but none showed virulence factors. No other yeasts or bacteria were isolated through mycological or bacteriological analysis. CPE was not observed in any of the oral swabs. Serological analyses did not detect antibodies specific to any of the investigated pathogens except for flaviviruses. Five serum samples tested positive in the ELISA test for WNV, but all tested negative in the confirmatory SN assays for WNV and USUV. Babesia sp., Borrelia sp., Leptospira sp., etc.) as well as for wild native fauna . The results suggested a potential role as a zoonotic carrier for the Eastern grey squirrel, supporting the importance of its management to guarantee human health. Moreover, information obtained by this management plan reinforces the surveillance of pathogens for humans but also for domestic animals and livestock  and geophilic  [T. mentagrophytes var. interdigitale (previously identified as T. mentagrophytes) has been previously reported in squirrels [p = 0.027) than in the other seasons, probably because of favourable climatic conditions, such as wet weather and more food availability. Moreover, the greatest number of dermatophyte-positive squirrels found in MU 28 could be presumably due to the overpopulation in this area, which represented the site of the release of the Eastern grey squirrel in Umbria. Despite their disease-free appearance, the Eastern grey squirrel was identified as a potential source of zoonoses due to a high percentage of dermatophyte-infected individuals (31.2%) found in 12 MUs . Moleculingense) . Accordiingense) . T. mentquirrels ,71. Dermquirrels ,74,75,76Borrelia lusitaniae and Coxiella burnetii detection also confirmed that the Eastern grey squirrel could represent a potential source of zoonoses, because as tick-borne disease agents, they carry ticks that occasionally bite humans. Tick samples previously collected from wild rodents in central Italy were positive for B. burgdoferi sensu latu and C. burnetii [B. lusitaniae positivity in squirrels could be related to dogs which came to the public park, especially in MU 26, as it is known that several species of ticks, such as Ixodes sp., are hosted by companion animals and wild animals, such as flying squirrels [C. burnetii detection could be attributed to contact with infected faeces and birth products [burnetii . The sququirrels . Howeverproducts . The Veterinary Police Regulation  and the information system of infectious and diffusive diseases  differentiate notifiable from non-notifiable diseases, providing useful management feedback. Lyme’s disease is not considered a notifiable disease—the management response was limited to the MU where the disease occurred, increasing the sample size for sanitary investigations. In contrast, dermatophytes and Q fever are defined as notifiable diseases; in these cases, both an individual-dependent response and an area-dependent response were applied. An individual-dependent response indicated an increase in the catch rate and restrictions for individual squirrel management. Activities such as the translocation of sterilised grey squirrels or the release of Eurasian red squirrels were also foreseen within the project. However, based on the results of the sanitary screening, no sterilised squirrels could be moved to/from the MUs where those pathogens existed, and neither could Eurasian red squirrels be released there (area-dependent response).No evidence of hantavirus was reported in the study area, whereas positive results from the serological test for flavivirus may indicate circulation of any flavivirus other than WNV and USUV among squirrels. C. albicans, their immune systems were probably compromised, but the low prevalence was not considered alarming for public health. This included the incidence of E. coli, which was not septicaemic or haemolytic, and was thus treated as commensal intestinal bacteria. Since all the swabs  collected from four animals tested positive for In particular, the authors noticed that all the analysed squirrels were found to be negative for the presence of SQPV DNA, establishing no evidence of its infection in Italy  and prevS. carolinensis and S. vulgaris. Naturally, the squirrel native species lives in balance with the local flora , whereas the non-native species is responsible for injuries in nuts’ harvesting and debarking of trees, resulting in severe economic losses. Moreover, due to their size, Eastern grey squirrels can prey upon nestlings, increasing biodiversity damage. In addition, their confident behaviour can be dangerous for humans, because they can be reservoirs of zoonoses such as dermatophytoses and tick-borne diseases. The LIFE U-SAVEREDS strategy could be suitable to achieve the right form of awareness about the previously discussed topics, in particular through environmental educational plans and scientific dissemination that involve the community. This survey on the Eastern grey squirrel population indicated that it is necessary to raise awareness of its impacts on biodiversity and human and animals’ health. The presence of this IAS in the Umbria region causes ecological damages through different processes, including competition, predation, parasitism, disease vectoring, hybridisation, and spillover/spillback mechanisms. In particular, the ‘exploitation competition’ mentioned previously is a complex process that occurs as a result of the concurrent presence of S. vulgaris is currently less life-threatened by S. carolinensis: the Eurasian red squirrel seems to have almost completely recovered its natural distribution area, as proven by the post-LIFE monitoring (data not yet published). Thanks to the eradication campaign, Other wild species could be investigated by similar methods to obtain interesting epidemiological data and adopt management advice to guarantee both ecological balance and human and animals’ health.


Rapid diagnosis and whole genome sequencing confirmed a case of monkeypox in an HIV-positive individual receiving antiretroviral therapy. The patient had a normal CD4+ T-cell count and suppressed HIV viral load and presented with a genital rash in Melbourne, Australia after return from Europe in May 2022. He subsequently developed systemic illness and disseminated rash and 11 days after symptom onset, he was hospitalised to manage painful bacterial cellulitis of the genital area. A rapidly emerging outbreak of monkeypox infection in over 20 countries from Europe, North and South America and the Middle East commenced in May 2022 . The maj3  and HIV viral load < 100 copies/mL, visited a primary care doctor after his return from Europe to Melbourne, Australia. He reported onset of a genital rash 8 days earlier. The rash had started 5 days after he reported unprotected insertive anal intercourse with four casual male partners in Europe. The initial symptoms were painless white pustules on the penis that became painful and pruritic. He reported that he developed a fever and malaise 3 days after the first appearance of the penile rash and over the subsequent 5 days, the rash disseminated to his trunk, then more sparingly to the face and limbs while the genital lesions crusted over.An HIV-positive man in his 30s receiving co-formulated Abacavir, Lamivudine and Dolutegravir and with a CD4 + T-cell count above 700 cells/mmOn the day of his initial visit to his primary care doctor, 8 days after symptom onset, he was treated presumptively for gonorrhea and chlamydia with intramuscular ceftriaxone and oral doxycycline. On day 10 after symptom onset, he was assessed again by his primary care doctor for increased genital pain and commenced oral cephalexin for superimposed bacterial cellulitis of his genitals. On day 11 after the onset of rash, his primary care doctor who was aware of a cluster of monkeypox cases reported in MSM in Europe from media reports, referred the patient for inpatient hospital care due to concern for monkeypox and for pain management .On admission to hospital, the patient was managed with contact and airborne precautions in a single room with negative pressure ventilation. These precautions were undertaken as the diagnosis was unclear and varicella zoster virus infection was a potential differential diagnosis requiring airborne precautions. When examined in hospital, the penile lesions had largely formed scabs and the lesions on hands and lower limbs were painless papular pustules. Lesions on the upper limbs and trunk were in various vesicular and crusted states . He was Chlamydia trachomatis and Neisseria gonorrhoea by standard methods using real-time PCR (RT-PCR) from urine and rectal and throat swabs, were all negative. Syphilis serology showed a rapid plasma reagin (RPR) titre of 8 and 4 when checked on two separate occasions, which represented over a 4-fold drop in RPR from when he was treated for syphilis 8 months prior, in 2021.Routine blood tests  performed when admitted to hospital were all within normal limits and a C-reactive protein was mildly elevated at 12 mg/L (reference range: 0–5 mg/L). Test results for varicella zoster virus, herpes simplex virus, Supplementary material). Whole genome sequencing performed as described in the Supplementary material of DNA derived from the skin lesions resulted in the complete recovery of the entire monkeypox genome  with phylogenetic analysis revealing clustering with other monkeypox virus sequences from the May 2022 outbreak in Europe and the United States (Swabs taken from deroofed skin lesions on the hand, calf and trunk in addition to combined nose throat swabs on the day of hospital admission, were all positive for monkeypox virus using previously described conventional  and in-hd States .In addition, monkeypox virus was isolated from swabs of lesions on the hand and abdomen in Vero/hSLAM cells, and the virus was visualised through thin section electron microscopy .Monkeypox is a zoonotic disease endemic to Central and West Africa and not historically associated with sexual contact ,4. ClassPotential sexual transmission is suggested by the initial presentation of penile lesions at the site where sexual contact occurred before the systemic illness. However, establishing if monkeypox can be sexually transmitted will require larger studies combining clinical, epidemiological, and detailed virological analyses including phylogeny of transmitted viral strains. Whether or not the HIV infection was contributory to the clinical course in the patient is unknown, but it may be possible that advanced or uncontrolled HIV infection could lead to more severe outcomes. Hospitalisation of our patient was not for severe manifestations of the monkeypox viral infection but to enable diagnostic evaluation, pain management and treatment of bacterial superinfection. His normal CD4+ T-cell count and supressed HIV viral load on antiretroviral therapy were potentially important factors in preventing more severe outcomes from his monkeypox infection.Monkeypox should be considered in the differential of a vesicular or pustular genital rash and requires prompt diagnosis and a public health response. This includes tracing close contacts and consideration of interventions such as isolation and post-exposure prophylaxis with smallpox vaccine if indicated.