Leucine-rich-repeat receptor-like kinases (LRR-RLKs) play central roles in sensing various signals to regulate plant development and environmental responses. The extracellular domains (ECDs) of plant LRR-RLKs contain LRR motifs, consisting of highly conserved residues and variable residues, and are responsible for ligand perception as a receptor or co-receptor. However, there are few comprehensive studies on the ECDs of LRR-RLKs due to the difficulty in effectively identifying the divergent LRR repeats.http://phytolrr.com/ for website usage and at http://github.com/phytolrr for local usage. The LRR-RLKs were classified into 18 subgroups (SGs) according to the maximum-likelihood phylogenetic analysis of kinase domains (KDs) of the sequences. Based on the database and the SGs, the characteristics of the LRR motifs in the ECDs of the LRR-RLKs were examined, such as the arrangement of the LRRs, the solvent accessibility, the variable residues, and the N-glycosylation sites, revealing a comprehensive profile of the plant LRR-RLK ectodomains.In the current study, an efficient LRR motif prediction program, the “Phyto-LRR prediction” program, was developed based on the position-specific scoring matrix algorithm (PSSM) with some optimizations. This program was trained by 16-residue plant-specific LRR-highly conserved segments (HCS) from LRR-RLKs of 17 represented land plant species and a database containing more than 55,000 predicted LRRs based on this program was constructed. Both the prediction tool and database are freely available at The “Phyto-LRR prediction” program is effective in predicting the LRR segments in plant LRR-RLKs, which, together with the database, will facilitate the exploration of plant LRR-RLKs functions. Based on the database, comprehensive sequential characteristics of the plant LRR-RLK ectodomains were profiled and analyzed.The online version contains supplementary material available at 10.1186/s12860-021-00344-y. Arabidopsis thaliana) LRR-RLKs   were identified and have been proved to be structurally imperfect but functionally competent mutants [Plant LRR-RLKs are important membrane-localized receptors sensing various ligands to regulate plant developmental processes. The classification of LRR-RLKs is usually based on the alignment of the KDs, because the alignment of the ECDs is too ambiguous for phylogenetic analysis. In this work, ~ 3000 LRR-RLK sequences from 17 represented land plant species were classified into 18 SGs based on the alignment of their KDs. The SGs in the ML tree agree with most of the reported classifications , 25, 26, segment , 18. Sev segment , 38, PSK segment , RGFR1 [ segment , HAE [30 segment , FLS2 [4 segment , and PEP segment , have be segment , S437 an segment , and G18 segment . Others, segment , which m mutants –46. Most mutants . Moreove mutants , 32, 33,Arabidopsis, based on the NxS/T (x ≠ P) motif, approximately ~ 1200 out of 4000 secretory glycoproteins contain more than five canonical N-glycosylation sites [+ in this work) tended to localized at the 5th, 8th, 10th, and 21st variable sites in the plant LRR consensus sequences  LRR(s) and a KD were extracted by running the hmmsearch (HMMER 3.2.1) program as described previously . The TMs Denmark . Protein Denmark . The obt Denmark  at http:https://www.uniprot.org/ and the NCBI at https://www.ncbi.nlm.nih.gov with manual verification. ClustalW alignment was carried out in MEGA 5.0 to get a snapshot of the highly conserved sequence segments [The Phyto-LRR prediction program was constructed based on the PSSM algorithm as described previously  with somsegments . A totalhttp://phytolrr.com), and those manually added into the database were listed in Tab. The plant LRR-RLK database (Phyto-LRR database) was created using MySQL. A total of 3987 non-redundant ECD sequences of plant LRR proteins from 17 plant species  were then determined based on the Phyto-LRR database. The soluble accessibility was predicted by ACCpro20 programs in the SCRATCH-1D suite [+ were also examined , PEPR1 (At1g73080), FLS2 (At5g46330), HAE (At4g28490), TDR/PXY At5g61480), PSKR1 (At2g02220), and RGFR1 At4g26540) protein sequences were used as queries to perform BLASTP search for their own homologous sequences in these 2999 LRR-RLK sequences  tree of SG_x, and ten test sets of the phylogenetic treeAdditional file 5: Table S4. N-glycosylation sites annotated in the Arabidopsis proteome GFF file from Swiss-ProtAdditional file 6: Table S5. Information on the protein sequence datasetsAdditional file 7: Table S6. Training dataset 1 for Phyto-LRR predict programAdditional file 8: Table S7. Training dataset 2 for Phyto-LRR predict programAdditional file 9: Table S8. The distribution pattern of LRR motifs and potential N-glycosylation sites of each LRR-RLK member in different SGsAdditional file 10: Table S9. Protein sequences blast from 17 plant genomes LRR-RLKs using Arabidopsis protein sequences as a query


Dear Editor,2. International Committee on Taxonomy of Viruses (ICTV) named the virus as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)3. SARS-COV-2 is transmitted mainly through respiratory droplets and close contact4. The fast spread of the coronavirus disease (COVID-19)3 suggests that SARS-COV-2 is highly contagious. The virus remained viable in the medium for 7 days at 22 °C and 1 day at 37 °C5. On dry surfaces at room temperature (RT), the virus was reported viable for 1 day on the surface of cloth, for 4 days on stainless steel, and for 7 days on the outer layer of a surgical mask, whereas no infectious virus was recovered from the surfaces of printing and tissue papers after a 3-h incubation5. Here, we first investigated the infectivity of SARS-COV-2 using a plaque-purified strain nCoV-SH01 isolated from a patient in Shanghai (GenBank MT121215)6, studied subsequently its stability in liquid medium, on dry filter paper, and under acidic condition (pH2.2) at RT. It would provide guidance on application appropriate measures to control the spread of SARS-COV-2 and improve laboratory biosafety management.The pneumonia caused by a novel coronavirus was first reported in December 2019 in Wuhan of China, and since then has become a pandemic50 assay (tissue culture infection dose), as 6 × 105 PFU/mL and 2.8 × 106 TCID50/mL, respectively. Cytopathic effects (CPE) appeared at 24 h post inoculation (h.p.i.) with 100–2000 PFU of the virus titer, at 48 h.p.i. with 5–50 PFU  virus in subsequent experiments.First the virus stock of nCoV-SH01 was quantified on Vero-E6 cells by plaque forming assay (plaque forming unit) and TCID4. How stable is the virus in different environment? We first determined viral stability in liquid medium. 1.2 × 103 PFU (3.75 Log10TCID50) virus in DMEM was added into each well kept in a wet box at RT. After set for 1, 2, 3, 4, 5, 6, or 7 days, respectively, 100 μL of the virus solution was transferred from each sample onto Vero-E6 monolayer. CPE were checked daily till day 5. We found that when the virus had been kept in the medium at RT for 1 day, CPE appeared at 24 h.p.i., which was like the untreated virus control. When the virus had been kept for 2 or 3 days, CPE emerged at 48 h.p.i. (Table 10TCID50). When the virus had been left in the medium for more than 4 days, no CPE was observed. The loss of infectivity was confirmed by TCID50 assay, immune florescence staining with the antiserum against viral N protein  virus in 5 μL DMEM was added onto sterilized filter paper in plates. After completely dried, the plates were put into a dry box at RT. After set for 1, 2, 3, 4, 5, 6, or 7 days, the virus on the filter paper was eluted with DMEM, respectively. The eluted virus titer was 3.42 Log10TCID50 after the virus remained on the paper air dried for 1 h (recovery efficiency was 103.42/103.75 = 10−0.33 = 46.77%) and CPE appeared on day 2 post inoculation. When the virus had been kept on dried filter paper for 1 or 2 days at RT, CPE appeared on day 4 or 5 post inoculation, respectively . For the 3-day incubation on dry filter paper at RT, although CPE was observed, the survived virus could not be quantified by TCID50 assay. The loss of virus viability in prolonged incubation (>4 days) was confirmed by N protein immunofluorescence staining, qRT-PCR, and further verified by blind passage of the supernatant for three generations. In Alex’s study, the virus remained viable in the medium for 7 days at 22 °C5. The reason for the longer survival time in their report might be the higher viral titer they used (~6.7 Log10TCID50 versus 3.75 Log10TCID50 in this study). Regardless, our results show that SARS-COV-2 is highly infectious and relatively stable in the environment, which underscores the importance of environmental disinfection and hand hygiene.SARS-COV-2 can be shed into wet or dry surrounding by droplets or aerosoli. Table . By day ly Table , and the9. Bioinformatics analysis of single-cell transcriptomes revealed that ACE2 was expressed in esophagus squamous epithelium cells and enterocytes of ileum and colon10, hence SARS-COV-2 might be potentially transmitted via the fecal-oral route12. We speculate that SARS-COV-2 would have to survive the gastric acidic environment if the virus is indeed transmitted via the fecal-oral route. Consequently, we determined the survival of SARS-COV-2 under acidic condition in vitro. Various amount of the virus  was treated with acidic physiological saline (pH 2.2) at RT for 30 s, 30 min or 60 min, respectively. After treatment, each viral sample was adjusted to pH 7.28 and added onto Vero-E6 monolayer. As shown in Table 3, 1.0 × 103, 5 × 102, or 1 × 102 PFU of the virus. CPE appeared at 72 or 96 h.p.i. with lower virus titers . No CPE was seen with 0.01 × 102 PFU virus while CPE from the same amount as the virus control (0.01 × 102 PFU) was readily observed at 72 h.p.i. After the 30-min or 60-min incubation in pH 2.2 saline, no CPE was observed with the virus titers equal and below 1.0 × 103 PFU, whereas CPE appeared with 1.2 × 103 PFU virus although the survived virus could not be determined by TCID50 assay  treated under the same acidic condition. The results suggest that SARS-COV-2 at a certain high titer might survive the acidic environment of the stomach for a certain period. Although it is unclear whether the virus can replicate in the intestine, the survived virus in the gastrointestinal tract may be excreted in faeces, which would indicate the importance of stool disinfection. Whether the virus can be transmitted through the fecal-oral route needs further study.Since some coronaviruses can cause infectious diseases of digestive tract via gastrointestinal transmission, such as mouse hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV), and feline enteric coronavirus (FECV)ay Table  and confay Table . TransmiIn conclusion, our findings show that SARS-COV-2 can survive for 3 days in liquid medium or on dry filter paper, and the virus at a high titer can survive under acidic condition that mimics the gastric environment. Our study would provide guidance on application of appropriate measures to control the spread of SARS-COV-2 and improve laboratory biosafety.Supplementary Materials, Table and Figures


Gaudreault et al.). On the zoonotic side, there is West Nile virus that was introduced into the United States in 1999 , by Golender et al., and the detection and characterization of BTV serotype 14 in Russia by Koltsov et al.. In 2006, BTV serotype 8 was introduced into Europe and caused substantial clinical disease and also transplacental virus transmission. The failure to remove BTV serotype 8 from bovine embryos with subsequent spread of the virus via embryo transfers demonstrated the need for screening of embryo donors and embryos for BTV in endemic areas . This is critical since embryo transfer techniques have been used as a way to safely transfer genetic materials from a BTV endemic area to non-endemic regions. Since 1999, 11 exotic BTV serotypes have been isolated in the Southeastern United States but only one has been found outside of the Southeastern region  that a long history of causing disease and economic losses. A description of the clinical disease associated with the incursion of BTV serotype 3 in Israel is provided by n region . An overAuerswald et al. demonstrates clear evidence of current circulation of these viruses in Cambodian domestic birds; these findings necessitate the need for increased surveillance for these viruses in this region. The factors affecting the risks of JEV introduction into the United States were assessed by Oliveira et al.; this was done by qualitative risk assessment. WNV emerged in Europe in the early 2000's and another flavivirus, Usutu virus (USUV), which was originally found in Africa, was identified for the first time in the 1996 in Italy. Vilibic-Cavlek et al. summarize the epidemiology of these two mosquito-borne flaviviruses (WNV and USUV) in Southern Europe. Entomological surveillance by Calzolari et al. demonstrates increased circulation of WNV in Northern Italy and the importance of temperature on WNV infection of mosquitoes. A less known flavivirus, Tembusu virus which causes an egg-drop syndrome in ducks, emerged in 2010 in China. Vaccines against Tembusu virus have been generated but understanding of the neutralizing immune response to infection or vaccination is minimal. Lv et al. describe the development of a plaque reduction neutralization titration assay to detect antibodies to Tembusu virus and found long-lasting neutralizing antibodies in sera from infected and vaccinated flocks. The Japanese serocomplex includes JEV, USUV and WNV; whereas, Tembusu is grouped within the Ntaya serocomplex. The Ntaya complex also includes the Bagaza/Israel turkey meningoencephalomyelitis virus. The development of a duplex RT-qPCR assay to detect and distinguish between the Japanese and Ntaya serocomplexes is reported by Elizalde et al.. Zika virus is the most recent emerging mosquito-borne virus for which there are limited animal models available. Ambagala et al. conducted experimental infections with Zika virus in cattle, chickens, pigs, sheep and chicken embryos. None of the animals were susceptible to experimental infection except for the chicken embryos; this could provide an additional tool for Zika virus investigations.Flaviviruses are discussed in the next series of manuscripts including Japanese encephalitis, Tembusu, Usutu, West Nile, and Zika viruses. Endemic circulation of Japanese encephalitis virus (JEV) in Southeast Asia is well-established; this is also an area where West Nile virus (WNV) is known to circulate. A seroepidemiolgical study by Kroeker et al. Although RVFV is primarily transmitted by mosquito bites it is known that it can also be transmitted by aerosol  which is a mosquito-borne zoonotic disease endemic in Sub-Saharan Africa. RVF is a significant public and animal health concern and reliable RVF virus (RVFV) infection models are badly needed to develop advanced diagnostic tools and control strategies. A review on presently available livestock models  for experimental RVF virus infections is provided by  aerosol . In addi aerosol . A compaeviously , 11; howAkhrymuk et al. discuss the development of this approach using Venezuelan equine encephalitis virus (VEEV), an often neglected but important zoonotic viral pathogen that can cause high mortality in horses.An important part of a successful mitigation strategy for emerging and endemic pathogens is the ability to detect the presence of the agent in a rapid, sensitive and specific manner. The final paper in this Special Edition describes a novel approach using nanoparticles to preserve arboviral RNA in blood samples of infected animals thus increasing the ability for detection. The emergence of a new viral disease could affect public and animal health as well as the economy of many countries worldwide as demonstrated by the current COVID-19 pandemic. Therefore, we cannot forget that other known and unknown animal and zoonotic pathogens could also pose considerable threats to animal and public health globally. Arthropod-borne pathogens have emerged and are re-emerging in increased numbers in the last decades. This Special Edition provides overviews of these arboviral threats and discusses current and novel strategies for their detection and control.WW, DM, and JR wrote the editorial with careful editing and comments from SZ and GS. All authors contributed to the article and approved the submitted version.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.


These findings underline the importance of appropriate handling of contaminated PPE during and following use in high-risk settings and provide interesting insight into the potential utility of cotton in limiting COVID-19 transmission.The spread of COVID-19 in healthcare settings is concerning, with healthcare workers representing a disproportionately high percentage of confirmed cases. Although SARS-CoV-2 virus has been found to persist on surfaces for a number of days, the extent and duration of fomites as a mode of transmission, particularly in healthcare settings, has not been fully characterized. To shed light on this critical matter, the present study provides the first comprehensive assessment of SARS-CoV-2 stability on experimentally contaminated personal protective equipment (PPE) widely used by healthcare workers and the general public. Persistence of viable virus was monitored over 21 days on eight different materials, including nitrile medical examination gloves, reinforced chemical resistant gloves, N-95 and N-100 particulate respirator masks, Tyvek, plastic, cotton, and stainless steel. Unlike previous reports, viable SARS-CoV-2 in the presence of a soil load persisted for up to 21 days on experimentally inoculated PPE, including materials from filtering facepiece respirators (N-95 and N-100 masks) and a plastic visor. Conversely, when applied to 100% cotton fabric, the virus underwent rapid degradation and became undetectable by TCID Similar trends were noted in the 2003 SARS outbreak, where healthcare workers accounted for approximately 20% of cases4.Since its emergence in China at the end of 2019, the unprecedented spread of SARS-CoV-2 has led to the declaration of a pandemic by the World Health Organization. The role of healthcare settings in the spread of this virus is of particular concern, as healthcare workers have represented a disproportionately high number of confirmed cases in the first months of the COVID-19 pandemic5. This in turn may increase the opportunity for viral contamination and subsequent spread. An understanding of the environmental stability and persistence of SARS-CoV-2 on contaminated PPE may have profound impacts on the handling of both reusable and single-use items during use and after wear.Among the many questions yet to be adequately addressed about the modes of transmission of COVID-19 are those related to fomite spread and environmental contamination. In healthcare settings in particular, numerous surfaces ranging from door handles to disposable gowns and gloves may become contaminated by droplets of infectious secretions and shed by infected persons. Further, global shortages in personal protective equipment (PPE) may result in items intended for single use being reused or worn for longer periods than recommendedThe present study aimed to determine the stability of SARS-CoV-2 on experimentally inoculated surfaces of PPE widely encountered by healthcare workers and the members of the general population. Our findings underline the importance of appropriate handling of contaminated PPE after its use and provide interesting insight into the potential utility of cotton masks in limiting COVID-19 transmission.hCoV-19/Canada/ON-VIDO-01/2020, GISAID accession# EPI_ISL_425177) were prepared in Vero E6 cells as previously described6. Endpoint titration of stock virus and all experimental eluates were calculated via the 50% Tissue Culture Infectious dose assay (TCID50) on Vero E6 cells in a 96 well plate format using the method of Reed and Muench7. As SARS-CoV-2 is classified as a Risk Group 3 pathogen, all experimental procedures took place in a high containment laboratory at the National Microbiology Laboratory in Winnipeg, Canada.Passage 3 stocks of SARS-CoV-2  prepared in a tripartite soil load as per ASTM standard9 containing mucin (Sigma #M3895), bovine serum albumin (Sigma #A1933) and tryptone (Sigma #T7293) to represent the organic components of typical virus-containing fluids shed by infected individuals10. Ten microliters of the resulting virus suspension were added via positive displacement pipette to the center of each coupon and left to air dry in a biosafety cabinet for 1 h. Once dry, coupons were placed in a vented plastic container and stored in a closed cabinet at ambient temperature for the duration of the study. Daily recordings of temperature and humidity at 12-h intervals were monitored with a Professional Thermo-Hygrometer with data logger (TFA Dostmann Product#30.3039.IT).Experimental inoculum consisted of stock virus  of each test material were sampled at the following time points post inoculation: T = 1 h (point of drying), 4 h, and at 1, 2, 3, 4, 7, 14 and 21 days. Two additional short-course studies on cotton were also included, using the same inoculation and sampling regime up to 3 dpi. Virus was recovered by elution of each coupon in 1 mL of culture medium (DMEM + 2% FBS + 1% Pen-Strep) with rigorous and repeated pipetting. Eluates were immediately tenfold serially diluted for endpoint titration in Vero E6 cells, with remaining volumes of eluates stored at − 80 °C for potential re-processing if required. Samples resulting in low-level cytopathic effect  underwent additional sub-passage to confirm the presence of viable virus. Additionally, on the first instance where all biological replicates of a given sample material resulted as negative, supernatants from the neat dilutions of inoculated Vero E6 cells underwent additional sub-passage to ensure capture of potentially low-level virus not visually detected on the first passage. At the completion of the study, the full remaining volume from each day 21 eluate was added to an individual well of a 6-well plate of Vero E6 cells and monitored for cytopathic effect to maximize viable virus detection.11. Thermal cycling conditions were 50 °C for 15 min for reverse transcription, followed by 95 °C for 20 s and then 40 cycles of 95 °C for 3 s, 60 °C for 30 s. Values are reported as log genome equivalents per mL based on CT values obtained with a standard curve of known concentration plasmid DNA encoding for the target nucleocapsid region.Real-time RT-PCR (qRT-PCR) was performed on samples collected at T = 1 h (point of drying), 4 h, and 1, 7, and 14 dpi to confirm successful elution from all surfaces and to assess differences in residual RNA compared to infectious particles. Briefly, supernatants were pooled from the three biological replicates of individual materials harvested at a given time point, and viral RNA was extracted with the Viral RNA Mini kit . The qRT-PCR was run on a LightCycler 96  using the EXPRESS One-Step Superscript RT-qPCR Universal Kit  with primers and probes targeting the nucleocapsid (NP) gene50 results, including averages and standard deviations, were performed using GraphPad Prism (version 7) software. Biological replicates with no recoverable virus were assigned a value of zero for the purposes of these calculations.Graphical representations of TCIDThe environmental persistence of SARS-CoV-2 dried onto surfaces of commonly used PPE and surfaces found in healthcare settings over a period of 21 days revealed dramatic differences in virus stability. By 4 h post-deposition on cotton, infectious SARS-CoV-2 virus was drastically diminished, and no longer quantifiable by 24 h. Conversely, while reduced almost to the limit of detection, SARS-CoV-2 remained viable for up to 21 days when dried on plastic and particulate respirator procedural masks. Real-time monitoring of ambient conditions revealed a steady temperature near 20 °C with 35–40% relative humidity.When a dried onto non-porous surfaces in an organic soil load SARS-CoV-2 showed sustained persistence for prolonged periods of time. Viable SARS-CoV-2 was recovered after 21 days on plastic, 14 days on stainless steel, 7 days on nitrile gloves and 4 days on chemical resistant gloves, though at significantly reduced levels compared to the initial inoculum . However, virus dried on this surface remained viable through the final sampling point at 21 days post-inoculation  remained on all three biological replicates  and N-100 (Mask 2) surface materials for up to 21 days. Decay patterns of virus viability were comparable on the two masks; both showing a nearly 1 log decrease from 24 to 48 h, stable titres between 2 and 4 days, and a steady decline from days 7 through 21  from the eluted coupon. At the time of inoculation, potential wicking of the inoculum from the cotton coupon onto the culture plate below was noted for that particular biological replicate, however this cannot be confirmed as the cause for maintained viability. No further positivity was observed at any subsequent sampling point.To provide additional confidence to stability data on cotton, two additional short-term persistence trials were carried out, with sampling at identical intervals as the initial long-term experiment but up to only 3 dpi Fig. . Results50 values  in health care settings. Disinfection of these devices has proven to be challenging20. Previous investigations by our group on the persistence of Ebola virus on PPE showed comparable trends, with viable virus decreasing by 99% within a single hour of deposition on cotton and becoming entirely undetectable by 24 h while RNA remained detectable throughout the course of study21. Similar to our results with SARS-CoV-2, the viability of SARS-CoV-1 when applied at 105 TCID50/ml was limited to 1 h on a cotton gown compared to 24 h on a disposable gown22. Although there was a slight decrease in the amount of RNA recovered from the cotton fabric when compared to the other materials, this was minor when compared to the substantial loss of virus viability (complete absence by 24 h in 8/9 replicates). While it is possible that retention of the RNA and virus in the cotton may have contributed to a small portion of the lowered recovery, clearly the greater than 6 logs reduction of viable virus over the PCR reflects the fact that cotton has a significant effect on virus viability. The porous nature of the cotton fabric, increasing the surface area onto which the inoculum droplet was adsorbed and subsequently subjected to drying, is likely to augmented viral degradation thereby decreasing virus viability as well as quantification of RNA. While this manuscript was in its revision stage, a study released by Riddell and colleagues corroborated our observations on the reduced viability of SARS-CoV-2 on cotton compared to other materials23. Using a similarly high titer inoculum, a significant loss of infectious virus was noted after an hour of drying, though persistence was detectable up to 7 days. This difference may be attributed to the specific material attributes of the cotton cloths used in the two studies, and/or differences in protein content in the concentrated virus preparation without use of a sucrose cushion.The extremely limited viability of SARS-CoV-2 on cotton aligns with survival results of other enveloped viruses on cotton, including those of human and avian origin24, others have questioned the efficacy of manufactured or home-made cloth masks to effectively prevent release of viral particles from infected individuals26. Our observations in the present study are not intended for inference on the ability of cotton masks to prevent aerosol transmission of SARS-CoV-2, nor do they address filtration efficacy of cotton against the virus. However, the fact that virus viability is rapidly reduced on cotton exposure has implications for droplet transmission for both the wearer and nearby contacts, and complements the growing support of widespread cotton mask use. Further, these results suggest that the use of cotton-based fabrics in healthcare settings may present a lower risk during handling for subsequent decontamination and reuse. The use of cotton mask covers to extend the usage of N95 masks), a growing trend in major medical centers27, presents an excellent example of such a risk mitigation strategy.The use of non-medical masks to limit the spread of transmissible respiratory diseases, including COVID-19, has been contentious. While mounting evidence suggests that simple cloth masks could be effective in limiting SARS-CoV-229. Our results are in agreement with those reported, however viral persistence was significantly prolonged under our experimental conditions. These discrepancies are likely due to a combination of different ambient conditions, inoculum titres, and readout assay sensitivity. Indeed, in the recently published work of Riddell and colleagues23, extended persistence of SARS-CoV-2 for up to 28 days was observed under experimental conditions most closely resembling those utilized herein.The survival of SARS-Cov-2 on environmental surfaces has been described in two heavily cited studies28, using a 5 µl droplet of high titre stock virus (~ 7.8 log TCID50 units/mL), most closely resembled our inoculation conditions. The reported 65% RH in their study compared to ~ 40% RH under our experimental conditions is likely a significant contributor to the reduction in viability over time. The detrimental effects of high relative humidity on survival of other coronaviruses, including MERS and SARS-CoV-1, has been demonstrated32. Nevertheless, the availability of data specific to diverse environmental conditions is of importance to capture the realities at different geographical locations.The work of Chin and colleagues29, viable SARS-CoV-2 persisted up to only 3 days on stainless steel and plastic. While the reported ambient conditions more closely represented ours, the titre of infectious SARS-CoV-2 applied to test surfaces was approximately 2 logs lower than that applied in our study. The protective effect of high titre inoculum on environmental persistence has been previously shown for SARS-CoV-122 and therefor the extended survival observed under our experimental design is not surprising.In the report by Van Doremalen et al.10 was included in our experimental inoculum. The added stability provided by matrices to viruses under drying conditions has been demonstrated for many viruses, including SARS-CoV-134. The recent findings of Riddell and colleagues, demonstrating persistence of viable SARS-CoV-2 up to 28 days on several surfaces23 strongly supports the role of an organic matrix combined with high titer inoculum in the extended viral persistence observed in our experimental setting.An additional factor differentiating the present work from other studies is the matrix in which inoculum was prepared. Contamination in a healthcare setting can occur through droplets from an array of infectious biological fluids, therefore a standardized tripartite soil load36. Clinical data from mild and severe cases has revealed a range of data, with median initial viral loads of 5.11 and 6.17 log10 copies of viral RNA per ml in mild and severe cases, respectively37. In a critically ill patient, loads of up to 1.34 × 1011 copies per mL were detected36. Data on infectious virus shed in excreta from acutely ill COVID-19 patients is lacking. However, in a recently published SARS-CoV-2 pathogenesis study, peak viral loads of > 6 log10 TCID50/mL, corresponding to 11 log10 TCID50 RNA copies per mL were observed in the golden hamster model38. Taking these data into consideration, our experimental model would more closely demonstrate environmental contamination from a severe COVID-19 case.The decision to use a high titre inoculum in our study was to represent a worst-case scenario of SARS-CoV-2 persistence on a contaminated surface. Shedding of viral RNA from COVID-19 patients varies widely based on sample type, time post-symptom onset, severity of disease, and on an individual basisA potential limitation of this study lies in testing being carried out in a high containment laboratory, where 12 air exchanges per hour may represent a high air flow not necessarily representative of most health facilities, homes and other environments. In addition, the initial hour of drying time under a biosafety cabinet would have accelerated the drying effect and may have shortened the amount of time to inactivate the virus. However, the revealed persistence of up to 21 days was significant and may be the best-case scenario associated with high airflow conditions that can occur in some health care environments.29 on the duration of potential fomite transmission of SARS-CoV-2 should be interpreted with caution due to the sampling methodology utilized. Viral recovery was achieved by material saturation in media, enabling recovery of extremely low levels of remaining virus. Had we employed a swabbing method to more closely mimic a casual contact scenario, a decrease in sensitivity would be expected and thus duration of viable virus detection may have been reduced. As a result, the data generated represent a worst-case scenario for potential exposure through these contaminated surfaces, though the risk of fomite transmission through casual contact can only be inferred by the presence of infectious virus at a given time point.Additionally, the implications of these results and those of othersOur findings suggest that SARS-CoV-2 can remain infectious on contaminated PPE for extended periods under ambient environmental conditions. Conversely, they demonstrate that stability is highly reduced on cotton even within hours of contamination, and encourage the use of 100% cotton in infection prevention and control practices where fluid-resistant barriers are not required. These results underline the importance of proper handling of personal protective equipment during and following use in high-risk settings to minimize the likelihood of fomite transmission, and may assist centers in developing guidelines and protocols relating to decontamination and reuse of PPE in short supply.Supplementary Information.


Ubiquitination of proteins is a post-translational modification process with many different cellular functions, including protein stability, immune signaling, antiviral functions and virus replication. While ubiquitination of viral proteins can be used by the host as a defense mechanism by destroying the incoming pathogen, viruses have adapted to take advantage of this cellular process. The ubiquitin system can be hijacked by viruses to enhance various steps of the replication cycle and increase pathogenesis. Emerging viruses, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), flaviviruses like Zika and dengue, as well as highly pathogenic viruses like Ebola and Nipah, have the ability to directly use the ubiquitination process to enhance their viral-replication cycle, and evade immune responses. Some of these mechanisms are conserved among different virus families, especially early during virus entry, providing an opportunity to develop broad-spectrum antivirals. Here, we discuss the mechanisms used by emergent viruses to exploit the host ubiquitin system, with the main focus on the role of ubiquitin in enhancing virus replication. Ubiquitin is a 76-amino acid protein well known for its function in marking proteins for degradation by the proteasome, although multiple non-degradative functions are well established. Covalent ubiquitination depends on a series of enzymes, including the E1 ubiquitin-activating enzyme, an E2 ubiquitin-conjugating enzyme, and an E3 ubiquitin ligase . UbiquitViruses have evolved to antagonize the host immune response by interfering with host ubiquitin-dependent signaling pathways, or by hijacking the cellular ubiquitination machinery, to promote viral replication and pathogenesis. With the ongoing pandemic caused by the novel strain of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), it is becoming critical to identify common steps used by different viruses to replicate in host cells that could potentially be targeted with broad-spectrum antivirals. Specifically, ubiquitination of viral proteins is emerging as an important host cellular process that is utilized by multiple viruses to promote their replication. In this review, we explore the various ways that emergent viruses exploit the host ubiquitin system, and we focus on recent literature highlighting the role of ubiquitin in enhancing virus replication .Viruses gain entry to the host cell through highly specific interactions with cellular surface receptors, although some viruses can also use less-specific mechanisms of entry via interactions with lipids or carbohydrates on the surface of the cell membrane . FollowiFurther evidence that flaviviruses in general may use a non-degradative ubiquitination step for uncoating of the virus genome comes from experiments with yellow fever virus (YFV). Similar to DENV, inhibition of the E1-activating enzyme also inhibited the initial round of YFV translation, and this required the function of the valosin-containing protein VCP/p97 , which i−/−Trim7 animals. A proportion of infectious viral particles released during replication contained ubiquitinated E, and ubiquitination on the E-K38 residue provided the virion the ability to interact with at least one potential cellular receptor, TIM-1, enhancing virus entry, replication and pathogenesis. In this case, ubiquitination of E not only functions in the early steps of virus entry, but also provides a mechanism of tissue tropism . Th. Th82]. The host E3-ubiquitin ligase TRIM6, along with the E2-ubiquitin conjugase Ube2K, synthesize unanchored K48-linked polyubiquitin chains that enhance the activation and kinase activity of IKKε . The unaOther henipaviral proteins block other steps of the IFN-I induction and signaling pathways. Similar to IAV and SARS- and MERS-CoVs, the accessory protein V (NiV-V) of NiV and other paramyxoviruses is capable of inhibiting TRIM25-dependent activation of RIG-I [To successfully replicate, viruses depend on specific factors expressed by the host cell. Many viruses use the ubiquitin system at different stages of their replication cycle, highlighting the importance of this cellular machinery for viral replication and spread. In this review, we provided an overview of the complexity of the interaction between emerging and highly pathogenic viruses such as SARS-CoV-2 and the ubiquitin system. Importantly, ubiquitination of structural proteins that are important in the process of virus entry is developing as a common non-degradative mechanism used by emergent viruses from different families, and could provide targets for broad-spectrum antivirals. One aspect that needs further investigation is how and when ubiquitinated proteins, or in some cases unanchored ubiquitin, are packaged in the infectious virions. Future studies should focus on identifying the specific steps and the subcellular compartments in which ubiquitination occurs to promote viral replication, and whether these processes are conserved through the different families of viruses. Identifying the specific factors of the host ubiquitin system that are commonly required for replication of different viruses could provide targets for broad-spectrum antivirals.


Alphamesonivirus, Quaranjavirus and unclassified Bunyavirales was detected in mosquitoes, and phylogenetic analyses revealed their similarities to viruses previously reported in other continents. To the best our knowledge, our findings constitute the first distribution record of these viruses in European mosquitoes and the first hint of insect-specific viruses in mosquitoes’ saliva in field conditions, demonstrating the feasibility of this approach to monitor the transmissible fraction of the mosquitoes’ virome. In conclusion, this pilot viromics study on honey-baited FTA cards was shown to be a valid approach for the detection of viruses circulating in mosquitoes, thereby setting up an alternative tool for arbovirus surveillance and control programs.Worldwide, emerging and re-emerging infectious diseases (EIDs) are a major burden on public and animal health. Arthropod vectors, with mosquitoes being the main contributors of global disease, transmit more than 70% of the recognized EIDs. To assess new alternatives for arthropod-borne viral diseases surveillance, and for the detection of new viruses, honey-baited Flinders Technology Associates (FTA) cards were used as sugar bait in mosquito traps during entomological surveys at the Llobregat River Delta . Next generation sequencing (NGS) metagenomics analysis was applied on honey-baited FTA cards, which had been exposed to field-captured mosquitoes to characterize their associated virome. Arthropod- and plant-infecting viruses governed the virome profile on FTA cards. Twelve near-complete viral genomes were successfully obtained, suggesting good quality preservation of viral RNAs. Mosquito pools linked to the FTA cards were screened for the detection of mosquito-associated viruses by specific RT-PCRs to confirm the presence of these viruses. The circulation of viruses related to  Worldwide, two-thirds of all recognized emerging and re-emerging infectious diseases (EIDs) are of viral origin , with arTraditionally, for active surveillance and control purposes, samples from entomological surveys and/or from sentinel animals are subjected to laboratorial analyses to evidence arbovirus circulation. Despite these methodologies being considered the “gold standards”, many issues must be considered. For instance, in entomological surveys, specialized personnel are required to capture and classify specimens, and a cold chain must be maintained to prevent virus degradation until molecular processing ,13. Due Since gold-standard strategies are time-consuming, logistically complex and potentially hazardous, honey-baited Flinders Technology Associates (FTA) cards have been used as an alternative tool for arbovirus surveillance as they inactivate pathogens and preserve nucleic acids on contact, thereby simplifying the labor ,13,16. ITo overcome the detection bias of molecular-based techniques, deep sequencing technologies have been proven as a valid approach to detect, characterize and discover unknown or uncultured viruses within biological or environmental samples ,21,22,23To the best of our knowledge, for the first time, next generation sequencing (NGS) on honey-impregnated FTA cards used as sugar bait during entomological surveys has been tested as a new approach for the detection of viruses circulating in mosquitoes. Viromics results on FTA cards were confirmed by the detection of mosquito-associated viruses in field-captured mosquitoes. Additionally, near-complete viral genomes were obtained. Herein, we show that insect-specific viruses (ISVs) can be detected in saliva from field-captured mosquitoes and report some ISVs previously identified in other continents, as first-distribution records in European mosquitoes.The present study was conducted at the Llobregat River Delta, North-Eastern Spain. In this Delta, densely populated areas coexist with natural habitats that serve as a strategic stopover on the route of migratory birds between Europe and Africa. For this reason, this area is considered to be of particular epidemiological interest and is targeted for arbovirus surveillance. In fact, sampling locations were chosen based on previous evidence of arbovirus circulation , and in 2-baited EVS Mosquito Traps . Inside the collection bag of some traps, one honey-soaked Classic FTATM card  [®  and coded according to location and sampling date. Only captured female mosquitoes were morphologically classified [Every fortnight, from May to November 2015, host-seeking female mosquitoes were captured using COire, UK) . At eachassified  and up tassified . FTA carPre-extraction, frozen FTA cards were thawed at 4 °C, homogenized with 500 µL of cold sterile PBS by vortex and squeezed with a sterile pestle to extract its content. Total RNA was obtained from individual FTA cards  using the RNeasy Mini Kit  according to the manufacturer’s instructions. Extracted RNA was eluted in 50 µL of RNase-free water. A unique RNA sample per biotope was generated by pooling 15 µL of all the corresponding extracts of the given area.RNA samples were sequenced and analyzed as previously described with slight modifications . Brieflyhttps://jgi.doe.gov/data-and-tools/bbtools) (cut-off parameter of 100). De novo assemblies were performed using Megahit tool [https://figshare.com/articles/translateReads_py/7588592), minimum aa length = 15), a Diamond-based similarity search (v0.9.22.123) against the protein Reference Viral database (RVDB-prot 16.0 [k-mer coverage of each contig.Raw reads were processed with an in-house bioinformatics pipeline as previously described . Summarihit tool  (minimumrot 16.0 ) was con® RNA Virus kit  following the manufacturer’s instructions. Using the OneStep RT-PCR kit , all the above-mentioned samples were screened for the detection of Alphamesonivirus 1, Bunyaviridae environmental sample, Dezidougou virus and Wuhan mosquito virus 7, adjusting the annealing temperatures to each set of primers  and with an identity higher than 90% were selected. Then, primers were designed from the extracted sequences of each chosen virus and conventional virus-specific reverse transcription polymerase chain reactions (RT-PCR) were set up. Viral RNA from mosquito pools and honey-baited FTA cards, which had not been exposed to mosquitoes, were then extracted using NucleoSpin primers . As posi® Gel Extraction Kit  and Sanger sequenced in both directions using the BigDye® Terminator v3.1 cycle Sequencing Kit . At the Servei de Genomica i Bioinformatica at the Universitat Autonoma de Barcelona (SGB-UAB), amplicons were purified with the BigDye X Terminator kit  and subjected to capillary electrophoresis in the Genetic Analyzer 3130xl . Viral sequences were aligned using BioEdit Sequence Alignment Editor [Culex bunyavirus/CAT virus. In both cases, a 1000 replicate bootstrap was used. Virus-specific RT-PCR products were purified using the QIAquickt Editor  and the t Editor . In the t Editor ,37 the bt Editor  with gamwww.ncbi.nlm.nih.gov/biosample/13978317 and www.ncbi.nlm.nih.gov/biosample/13978318). All the viral genomes for which the complete CDS were obtained were deposited in the GenBank archive under the accession numbers: MT096515-MT096531. Sequences corresponding to the viruses detected in mosquito pools from the Llobregat River Delta are available under the accession numbers: MT063093-MT063099. The raw sequencing datasets for both batches of honey-baited FTA cards are available in the NCBI Sequence Read Archive (SRA) repository under the BioProject ID: PRJNA604676 , Coquillettidia richiardii , Culex pipiens , Aedes caspius  and Aedes detritus   . A totaldetritus . Next generation sequencing (NGS) on honey-baited FTA cards generated 61,362,209 and 80,631,320 of raw reads for rural and peri-urban biotopes, respectively. After filtering steps, 56,424,764 and 76,884,845 reads of 150 bases were assembled to produce 431,179 and 100,469 contigs respectively for rural and peri-urban datasets. Depurated reads also generated 3,128,224 and 846,017 singletons in each case. Picornavirales, Nidovirales and Tymovirales were the most represented single-stranded positive sense RNA (ssRNA+) viral orders; and Bunyavirales the most abundant single-stranded negative sense RNA (ssRNA-) order. Double-stranded RNA (dsRNA) viral families Partitiviridae and Totiviridae were also dominant. DNA and unclassified viruses comprised the remaining 5% of the viral diversity herein reported  were mostly related to hymenopterans, in particular to the honeybee Apis mellifera. Since we could not sequence the honey used to impregnate the FTA cards as sugar bait, we cannot discard the possibility that these sequences might have come from it. However, recent virome studies have described these two families as the most abundant in culicid mosquitoes from the Yunnan province in China, and Zambezi province in Mozambique [Rhopalosiphum padi virus  in mosquito species from Hubei, China [Culex mosquitoes from California [Apis mellifera filamentous virus (dsDNA Hytrosaviridae family) from French Anopheles maculipennis [Culex mosquitoes from California [In the present study, taxonomic profiling revealed the prevalence of invertebrate-associated viruses A with Difamilies B. Sequenzambique ,50. The i, China  and in Clifornia , togethelipennis  and fromlifornia  respectilifornia ,50,53,54Tymoviridae, Totiviridae, Partitiviridae, Endornaviridae or Virgaviridae) as part of the viral diversity associated to honey-baited FTA cards  that was isolated from a Culex spp. pool from Xinjiang (China) was also shown to produce a cytopathic effect on Aedes albopictus C6/36 cell line [Besides invertebrate-related viruses, it was not surprising to find viral families usually detected in plants, fungi and algae  identity) and Calbertado virus (47–86% aa identities) (Phenuiviridae were assigned to a distant Phasi Charoen-like phasivirus with aa identities ranging from 58% to 77% (Peribunyaviridae-associated sequences presented high homologies with Ganda bee virus (35–95% aa identity)  virus families: ntities) . Reads r% to 77% . Meanwhidentity) . FinallyNidovirales (Alphamesonivirus 1: Ngewotan virus) and Picornavirales , and within unclassified RNA viruses  were generated  for which the 5′ and 3′ termini are incomplete since RACE-PCRs were not performed. Viral genomes within the orders enerated . ObtainiBunyaviridae environmental sample (335.292 abundance in nts) and Wuhan mosquito virus 7 (43.351 abundance in nts) were selected to design specific primers and set up virus-specific RT-PCRs. All these selected viruses showed to have at least one contig with a matching sequence longer than 1000 nt and similarity above 90%. For identification matters, through the manuscript, these viruses would respectively be referred to as Alphamesonivirus/CAT virus, Culex bunyavirus/CAT virus, Dezidougou/CAT virus and Wuhan mosquito/CAT virus. Suffix “CAT” stands for the geographic region of detection, i.e., Catalonia. To confirm virome results obtained through metagenomics analysis on honey-baited FTA cards, among all the mosquito-associated viruses , AlphameCulex bunyavirus/CAT virus and Alphamesonivirus/CAT virus, and positive for Dezidougou/CAT virus and Wuhan mosquito/CAT virus. These detections could be explained by (i) the presence of non-culicid dipterans in the traps; they could have deposited these viruses while sugar feeding from the FTA cards, and/or (ii) the source of these viruses came from the honey impregnated on the cards. Those honey-baited FTA cards, which were not exposed to mosquitoes recovered from entomological surveys, were then screened individually by virus-specific RT-PCRs to verify the source of the viruses detected by viromics. Screenings of both cards tested negative for Culex bunyavirus/CAT virus  was the most common and was recurrently detected in both biotopes , showing a high occurrence of this viral strain in Cx. pipiens mosquitoes from the Llobregat River Delta. BLASTn analysis of the amplified fragment of a RT-PCR positive pool showed a nucleotide similarity of 97.58% to Bunyaviridae environmental sample’s RNA-dependent RNA polymerase gene (RdRp). Phylogenetically, our strain clustered with Bunyaviridae environmental sample (2013) and Culex Bunyavirus 2 (2016), which have previously been detected in Culex spp. mosquitoes from the United States of America (USA)  A. The dilifornia ,57 and Mlifornia , to SpaiCx. pipiens pools, two Cq. richiardii and one Ae. caspius pools. Alphamesonivirus is the only recognized genus within the mosquito-restricted family Mesoniviridae  [Culex spp. mosquitoes. Houston virus (HOUV) and Nam Dinh virus (NDiV) in Culex quinquefasciatus from Mexico and China; Ngewotan virus in Culex australicus from Australia; NDiV, Alphamesonivirus-1 and HOUV in Culex spp. from China, South Korea, and the USA. It is worth mentioning that the viral strains detected in the present study demonstrated a closer relationship to each other than to the strains found in other geographic regions. Moreover, Alphamesonivirus/CAT strains found in Cx. pipiens, both rural and peri-urban biotopes, appeared to be more closely related to each other than to those found in other mosquito species from the same geographic area  . Strainshic area B, thereb, France , confirmiviridae . Recentliviridae ,49,53,61Cx. pipiens pools , which has been identified in a pool of Anopheles sinensis and Culex quinquefasciatus mosquitoes originating from Hubei, China [Ae. albopictus nor Ae. detritus were found to be infected by any of those viruses targeted. Detecting Culex bunyavirus/CAT and Wuhan mosquito/CAT viruses in Cx. pipiens pools, which were not exposed to honey-baited FTA cards, evidenced that these viruses were indeed infecting the mosquitoes and were not acquired while sugar feeding on the FTA cards.Finally, Wuhan mosquito/CAT virus was positively detected in six of 53 ns pools . Among t in 2013 C. Wuhan i, China . FinallyCulex bunyavirus/CAT, Alphamesonivirus/CAT and Wuhan mosquito/CAT viruses in culicid mosquitoes found in Catalonia, contributes to the knowledge of both the host range and their geographical distribution.The discovery of Culex bunyavirus/CAT virus, Alphamesonivirus/CAT virus and Wuhan mosquito/CAT virus in mosquito pools confirmed the presence of these viruses in Europe, where previously their circulation had not been revealed. These findings highlight the value of honey-baited FTA cards combined with viromics in identifying a wide spectrum of viruses that may be associated to sylvan mosquitoes in susceptible areas for arbovirus transmission, without requiring previous knowledge of viral diversity. In future arbovirus surveillance, NGS on honey-baited FTA cards could be used as a guide for prevention and control strategies. In the case of arboviruses detection, entomological surveillance could be exhaustively carried out focusing on specimen classification and molecular analysis where the virus of interest has been previously detected in the FTA cards.The current study is a pioneer in applying viromics on honey-baited FTA cards during entomological surveys as a tool for the detection of circulating viruses in mosquitoes and the identification of virus in mosquitoes’ saliva. Through this approach, 19 ssRNA (+), six ssRNA (−), eight dsRNA, one ssDNA, five dsDNA viruses and several unclassified viruses were identified; and 12 near-complete viral genomes were obtained from FTA cards, among which seven were linked to mosquito species of sanitary relevance. Acquiring near-complete virus genomes is a clear advantage of metagenomics over classical surveillance based on PCR detection, since insights into the origin, evolution, and diversity of circulating viruses could be gained . FurtherAedes aegypti despite the fact that there not been any record of blue dye in their abdomens [It is worth mentioning that, in spite of the advantages provided by NGS on honey-baited FTA cards, there are some drawbacks that need to be mentioned. Firstly, since FTA cards inactivate the viruses, and NGS provides only genetic information through this approach, no viable virus could be isolated for further characterization. Secondly, virus-bearing mosquito species could not be identified without complementary morphological and molecular analyses. Other possible constraints of this approach could be related to the feeding rate on FTA cards, the quantity of saliva expectorated by mosquitoes, and the number of viral copies liberated within the saliva while sugar feeding. The assumption of blue abdomens in mosquitoes, as the only proof of virus expectoration on FTA cards, might possibly overlook virus release while probing. This fact was evidenced with the detection of chikungunya virus (CHIKV) RNA in FTA cards exposed to experimentally infected abdomens . Based oabdomens . Culex flavivirus (family Flaviviridae) and Phasi Charoen-like virus (PCLV) , as they were also detected in salivary glands of Cx. pipiens from Iowa [Ae. aegypti from South China [Aedes flavivirus RNA in saliva from colonized Ae. albopictus [The detection of ISVs through metagenomics on honey-baited FTA cards provides evidence that these viruses could be transmitted within mosquitoes’ expectorations, thereby contradicting previous beliefs that they could not be expelled with saliva . Our finrom Iowa  and in Ath China , respectbopictus . Sequencbopictus ,66. Howebopictus . FurtherCulex flavivirus naturally infecting Cx. pipiens from Colorado possibly suppressed the early infection with West Nile virus (WNV) [Aedes aegypti were reduced by intrathoracic inoculation of newly isolated cell fusing agent virus (CFAV) [Aedes vexans form Catalonia seemed to decrease the susceptibility of infection to Rift Valley fever phlebovirus (RVFV) following experimental oral exposure [Furthermore, ISVs are a significant part of the mosquito’s virome. Due to their phylogenetic relationships, great abundance and high diversity, it is presumed that arboviruses might have been originated from arthropod-infecting viruses ,68,69. Ius (WNV) . In Thais (CFAV) . Likewisexposure . Culex bunyavirus/CAT and Wuhan mosquito/CAT viruses so as to assess their potential as vertebrate pathogens. Finding these ISVs in FTA cards, and therefore in mosquitoes saliva, rises concerns of the potential of these viruses to evolve from being insect-specific to dual-host viruses, acquiring the ability to infect vertebrate cells and become new emerging pathogens. Future surveillance strategies for emerging diseases could include NGS on honey-baited FTA cards to detect previously undiscovered and potentially transmissible viruses so as to prevent new arbovirus outbreaks. As evidenced, and in spite of the continual discovery of novel mosquito-associated viruses, viral diversity harbored by vector species is still underestimated and little is known about their host range, distribution, ecology and evolution ,73. FurtAlphamesonivirus, Quaranjavirus (Wuhan mosquito virus), and unclassified Bunyavirales in European field-captured mosquitoes using virus-specific primers derived from metagenomics results, demonstrated that viromics on honey-baited FTA cards is a valid approach for virological surveillance in mosquitoes. To the best of our knowledge, this is the first evidence of circulating ISVs in mosquitoes’ saliva under field conditions. Our study also constitutes the first distribution record of these viruses in the European continent, thereby demonstrating that they are widely distributed despite there being an information gap due to the majority of studies being focused primarily on arbovirus detection. Further studies are needed to better understand the evolutionary history of insect-specific viruses and their potential role in arbovirus transmission. The detection of viruses related to


Influenza is a major cause of mortality and morbidity worldwide. Despite numerous studies of the pathogenesis of influenza in humans and animal models the dynamics of infection and transmission in individual hosts remain poorly characterized. In this study, we experimentally modelled transmission using the H1N1pdm09 influenza A virus in pigs, which are considered a good model for influenza infection in humans. Using an experimental design that allowed us to observe individual transmission events occurring within an 18-hr period, we quantified the relationships between infectiousness, shed virus titre and antibody titre. Transmission event was observed on 60% of occasions when virus was detected in donor pig nasal swabs and transmission was more likely when donor pigs shed more virus. This led to the true infectious period (mean 3.9 days) being slightly shorter than that predicted by detection of virus (mean 4.5 days). The generation time of infection (which determines the rate of epidemic spread) was estimated for the first time in pigs at a mean of 4.6 days. We also found that the latent period of the contact pig was longer when they had been exposed to smaller amount of shed virus. Our study provides quantitative information on the time lines of infection and the dynamics of transmission that are key parts of the evidence base needed to understand the spread of influenza viruses though animal populations and, potentially, in humans. Influenza is a major cause of mortality and morbidity worldwide. The relationship between the time course of influenza infection and virus shedding and onward transmission of the virus remains poorly characterized. Pigs are a natural host for influenza infection with shedding patterns similar to humans. Therefore we experimentally infected pigs with the H1N1pdm09 influenza A virus using direct contact challenge and then mixed the infected pigs with a different naïve pig each day to understand when transmission occurred. Using mathematical modeling, we found that transmission events occurred on 60% of occasions when the infected pigs were shedding virus and that the risk of transmission increased with the quantity of virus shed. Also it was clear the incontact pigs started to shed virus later after exposure when the infected pigs were shedding low quantities of virus. Our study therefore provides quantitative information on the time lines of influenza virus infection and the dynamics of transmission. This is important to understand the spread of influenza viruses through animal populations and, potentially, in humans. The 2009 H1N1 pandemic demonstrated the potential of swine origin influenza viruses to cause significant human morbidity and mortality globally . Swine iUnderstanding SwIV transmission is pivotal to managing the risk of spread between pigs and spillover to humans. However, despite the common occurrence of H1N1pdm09 in pigs there is limited information on the viral kinetics and parameters determining influenza A virus transmission between pigs. Transmission models used to predict epidemics rely on natural history parameters that describe the time-course of influenza, such as the latent period or the duration of infectiousness. In addition, it has been previously assumed that infectiousness  is proportional to viral titre. However, this hypothesis has never been tested to our knowledge. It is therefore crucial to provide estimates of natural history parameters and to investigate the link between shed virus titre and the probability of transmission.Because the pig is an important natural host for influenza A viruses and a source of new pandemic viruses, it is also an appropriate large animal model for human influenza. Pigs and humans are infected by the same subtypes of virus and have similar distributions of sialic acid receptors in the respiratory tract ,10. The Here, we used an experimental design in which experimental SwIV transmission was analyzed in pigs infected by contact. In our study, a series of individual recipient pigs were placed in contact for a short time with a donor pig also infected by contact. This allows us to follow the time course of a contact acquired influenza virus infection including, precise information on when an infected donor is infectious to a susceptible recipient. We analyzed data on viral and antibody titres to identify determinants of transmission between pigs and to quantify the infection kinetics of naturally acquired H1N1pdm09 influenza in pigs.Twelve individual transmission experiments were performed. During each experiment, two “seeder” (S) pigs were intranasally inoculated with A/swine/England/1353/2009 virus (H1N1pmd09). Two days post inoculation (dpi), the two S pigs were put in contact with two “donor” (D) pigs for 24 hr to allow natural infection. After the 24 h exposure, one D pig was chosen randomly, designated as donor 1 (D1), and placed in contact with a different naïve recipient (R) pig each day for 18 hours during an 8 day period (recipients R1 to R8) . The secWe recorded a transmission event when a R pig shed virus above the limit of detection at least once. All donor pigs transmitted influenza virus at least once during the 8 days of follow-up  & S2 FigThe average daily shed virus titre was above the limit of detection in D pigs for 67/88 D-R contacts. Among these 67 contacts where the D pig was shedding virus, transmission to R pigs occurred on 42/67 (60%) occasions. Only one R pig was infected after being in contact with D pigs with undetected virus shedding: 5R1 (before the onset of detected shedding)  and 4A. When virus is detected, transmission events occur more frequently when antibody titre is ≤1:10 than when antibody titre is >1:10 at the first time of contact with 39 out of 56 (69.6%) vs. 1 out of 11 (9.1%) transmission events respectively. When virus is not detected, only one out of 10 (10%) transmission event was observed when antibody titre ≤1:10 and none when antibody titre > 1:10.We fit a Bayesian regression model to estimate the effect of virus and animal properties on the probability of transmission. The best model fits included the following standardized predictors: the average raw shed virus titre of the D pig during the contact period, the D pig antibody titre at the beginning of the contact period, the D and R pigs’ breed and R pig age at time of first contact . The modParameters estimates are shown as odds in D pigs’ level of antibody titres, on the other hand, tended to decrease the odds of transmitting influenza virus (odds = 0.171). However the 95% credible interval contained the value 1 (95%CI = [0.002–1.92]). Similarly for breed, Landrace pigs tended to decrease the odds of transmitting influenza virus (odds = 0.555). However the 95% credible interval contained the value 1 (95%CI = [0.138 − 1.92])  & Fig 6.A, increasing slope, s1A, time from infectious contact to the maximal shed virus titre, Tmax and decreasing slope, s2A) and “plateau” . We did not make any assumption of the individual viral kinetics profiles. The proportion of pigs showing a peaked pattern, p, was unknown and the group to which each pig belongs was defined as the group of highest conditional probability.We fit a viral kinetics model to D pigs viral shedding titres. We have tried to fit previously developed mechanistic models ,16–18, hA, LB, Tmax, T1, and T2. 1A = 2.17 log10(pfu/mL)/day or s1B = 1.16 log10(pfu/mL)/day until Tmax = 4.25 dpc or T1 = 2.70 dpc, for peaked and plateau, respectively. Shed virus titre decreased with slope s2A = 0.798 log10(pfu/mL)/day after Tmax for peaked responses or with slope s2B = 1.48 log10(pfu/mL)/day after a plateau lasting 2.78 days until T2 = 5.48 dpc  were only observed in D pigs. An average maximal shed virus titre Vmax = 4.58 ± 0.29 log10 pfu/ml was observed at Tmax = 4.51 ± 0.32 dpc in D pigs. Virus was detected for the last time at 7.05 ± 0.28 dpc on average leading to an average duration of shedding of 4.51 ± 0.31 days. The resulting area under the curve (AUC) was estimated as 17.1 ± 1.5 log10 pfu/ml. However it is noteworthy that the duration of shedding was right-censored since several pigs did not reach undetectable virus titre at the end of follow up (max in contact pigs (i.e. D pig in S-D contact and R pig in D-R contact) were significantly associated with the daily average shed virus titre. Pigs exposed to higher shed virus titres started to shed virus earlier and shed on average higher shed virus titres , the average antibody titre was 1:120.5 ± 1:82.8.To assess the accuracy of the model predictions, we simulated viral and antibody kinetics over 100 replicates of the original dataset and computed the relative bias between the natural history parameters computed from the original dataset and the simulated datasets.For all natural history parameters, the relative bias varied between -20% and +20%  & Fig 7.Experimental transmission studies can provide valuable parameter estimates to inform models of disease spread in animal populations. However, the reliability of these transmission studies depends on the animals transmitting virus receiving a challenge consistent with natural exposure in the field. We established a one-to-one transmission model to accurately estimate transmission parameters of H1N1pdm09 virus in pigs. Donor animals were infected by contact with a seeder pig to mimic naturally acquired infection, with the time of infectious contact known. These donor animals were closely monitored with nasal swabs taken twice daily (before and after contact with the recipient) for shed virus titre measurement and blood samples taken daily for antibody titre measurement. Donor animals were in contact for an 18h period with one recipient pig to allow identification of transmission events and avoid an extended exposure window for the recipient animal.We found that seeder pigs were shedding virus at very high titers shortly (<24h) after inoculation, while donor pigs started to shed virus between 1 and 6 dpi/dpc which is consistent with experiments where contact pigs were co-housed with animals inoculated with A/swine/Cotes d’Armor/0388/2009 H1avN1 (clade 1C) strain which started to shed virus between 3 and 7 days ,21.Consequently, the observed time to viral titre peak was also observed later in D pigs than in human experimental infections where it consistently averaged at 2 days post inoculation ,16,21–23The duration of shedding (4.51±0.31 days) was similar with experiments where 10 unvaccinated contact pigs were co-housed with one pig inoculated with A/swine/Cotes d’Armor/0388/2009 H1avN1 shed virus during 4.50±1.07 days  but was Overall, compared with seasonal A/H1N1 virus experimental infection studies in humans, during which inoculation was most frequently intranasal with the strain A/Texas/36/91 ,16,21–23All these results, suggested that high dose exposure would reduce the influenza latent period. We could not compare the dynamics from human experimental challenge with the S pigs’ influenza dynamics that also received intranasal inoculum because of their short follow-up (2 days).Virus shedding was, as expected, by far the most important predictor of transmission events. However, despite using an experimental set-up designed to maximize the chances of transmission over an 18 hour period, we only observed transmission on 60% of occasions when virus was detected in the D pig. We note that, importantly, estimates of the transmission period are slightly shorter than the duration of shedding . This diWe fit a mixture of segmented linear models with random effects to describe the donor pigs viral kinetics and variability. We considered two viral shedding profiles: peaked (47.5% of donor pigs) and plateau (52.5% of donor pigs). Pigs shedding virus according to the plateau pattern shed more, earlier and for a longer period.We tried and did not succeed to fit mechanistic models as previously used in human, mice and ponies ,16–18, uRecipient pigs exposed to high shed virus titres from donor pigs started to shed virus earlier and at a higher level and therefore had a higher probability of transmitting infection. We also found that younger pigs exposed to higher virus titre during the contact period were at higher risk of being infected. We estimated influenza generation time for the first time in pigs at 4.6 days (with low variance). We note that estimated generation time varies slightly with the scale used to quantify virus titre.Epidemic models rely on infection kinetics parameters . We therOne limitation of our study was that we did not control for the behavior of the donor and contact pigs, though this may have influenced the likelihood of transmission. Our study probably underestimates variability under natural conditions where animals of different ages, socialisation and immune status are co-housed. Also, we cannot determine whether direct contact, aerosols or contact with contaminated surfaces was the most important mode of transmission in our experiments ,27.There is a critical need to develop better models to understand influenza A dynamics that more closely mimic events in humans. Pigs and humans are infected by the same subtypes of influenza A viruses and integrally connected in the ecology of influenza A viruses . Pigs haTo our knowledge, this is the first experimental design of one to one transmission using in-contact infected pigs which allowed us to analyze both viral kinetics and transmission events from animals with naturally acquired influenza virus infection. This allowed us to estimate key parameters including the latent period, duration of virus shedding and onset of antibody production and, to relate these to direct observation of precisely timed transmission events. The design generates much more precise data than can be obtained from the observation of natural transmission events, as in studies of influenza virus transmission in humans . In addiAnimal experimentation was approved by the Pirbright Institute Ethical Review Board under the authority of a Home Office project licence (70/7505) in accordance with UK Home Office Guidance on the Operation of the Animals (Scientific Procedures) Act 1986 and associated guidelines.Twelve transmission experiments were performed. Female pigs were used between seven and ten weeks of age (except for transmission 4 where the pigs were 11 weeks and transmission 5 where they were of unknown age). All pigs weighed 18 to 40 kg, were either Landrace or Landrace x Hampshire breed and were obtained from a commercial high health status herd. All pigs were acclimatized for a period of 7 days before the start of the transmission experiment and randomly allocated to become seeders, donors or recipients.7 pfu of MDCK grown A/swine/England/1353/2009 virus (H1N1pmd09), as previously described [Two “seeder” (S) pigs were intranasally inoculated with a mucosal atomization device (MAD) with 1 x10Nasal swabs were collected daily for the S and R pigs and twice daily (at 8 am and 2 pm) from D pigs with the exception of transmission 1, where only the 8 am nasal swabs were taken. Two nasal swabs (one per nostril) were placed into 2 ml of virus transport medium comprising tissue culture medium 199 (Sigma-Aldrich) supplemented with 25mM Hepes, 0.035% sodium bicarbonate, 0.5% BSA, penicillin, streptomycin and nystatin, vortexed, centrifuged to remove debris and stored at -80°C for subsequent virus determination.Infectious shed virus titres in nasal swabs were determined by plaque assay on MDCK cells. Samples were 10-fold serially diluted and 100 μl of each dilution added to confluent MDCK cells in 12 or 24 well tissue culture plates. After 1 hr, the plates were washed and overlayed with 2 ml 1:3 2% agarose: medium. Plates were incubated at 37°C for 48 hrs and plaques visualized using 0.1% crystal violet.Blood samples were collected daily from the 24 D pigs and were not collected from S or R pigs. The microneutralization assay was carried out as previously described with the following modifications . Pig serWe used a Bayesian logistic regression to quantify the effect of each putative predictor on DR transmission events. We included the following putative population predictors: average shed virus titre  of the D pig during the contact period, D pig antibody titre at the beginning of the contact period, D and R pigs’ breed, R pig age at time of first contact.Inputs were standardized following Gelman’s recommendations ,33. The 1 and A2 representing the time to antibody production and the slope of increasing antibody titre, respectively.To model antibody kinetics, we used a segmented regression with mixed effects described by 2 parameters, A10- and log2-transformed for subsequent analysis, respectively. To model viral kinetics, we used a mixture of segmented regressions with mixed effects. We call zi the label of the structural model for pig i, with M1 for “peaked” (M1) and M2 for “plateau”. In the peaked model, after a latent period lasting LA, shed virus titres increase from below the limit of detection to the maximum shed virus titre Vmax occurring at time Tmax according to the slope s1A and then decreases according to a slope s2A. In the plateau model, after a latent period lasting LB, shed virus titre increases from below the limit of detection to the maximum shed virus titre Vmax according to the slope s1B. Shed virus titre remains at Vmax during the plateau between T1 and T2, and then decreases according to a slope s2B.Influenza shed virus titre (in pfu/ml) and antibody titre were logμ), the inter-individual variance-covariance matrix (Ω), the variances  of the additive error terms for viral kinetics (VK) and antibody kinetics (AK) models and the vector π =  of probabilities for each virus shedding profile (M1 or M2 here). The random effects and error terms were assumed to follow θ =  the vector of population parameters.We estimated the vector of fixed effects  algorithm implemented in Monolix2018R1 . In a fiWe considered that pigs were infected if virus was detected from the nasal swab. In D pigs this was confirmed by the detection of antibody at the end of the experiment.max), time to Vmax, onset of transmission, generation time, proportion of exposed contacts who becomes infected [The natural history of a disease refers to the time course of a disease in the absence of treatment and more specifically to the latent period/incubation, duration of shedding, maximal shed virus titre  pigs. We therefore defined the onset of transmission as the time interval between the infection of an individual and the first time when a transmission event is observed from this individual. Here, the onset of transmission and duration of transmission are upper and lower limit values respectively.Tg is the period of time between the onset of virus shedding in a primary case and the onset of infectiousness in a secondary case infected by the primary. It has been assumed that the probability to observe a transmission from a D to an R pig at time t, also called infectiousness is proportional to D1 pig’s viral shedding at t, V(t) [k1 the proportionality constant. We also assumed random contacts between contact donor (D1) and R pigs . Hence, the probability of observing a transmission event at time t is given by: P(E = 1│t) = k1k2V(t).Generation time,  t, V(t) ,41. We cTg as the expectation of P(E = 1│t) by integrating P(E = 1│t)t with respect to t and normalized by the total rate of transmissions over time, which is k1k2AUC. Dropping the constants k1 and k2 leads to the following expression:The total amount of virus shed by a D pig is given by the area under the curve, All integrals were computed using the trapezoidal rule as implemented in the caTools package . We presTo assess the accuracy of the model predictions, we simulated viral and antibody kinetics over 10 days for 100 datasets of 11 pigs, using the mlxR package. Individual parameters were sampled from the estimated population distribution.Using the viral kinetic profiles simulated for the 11 pigs in each replicated dataset, we simulated the transmission events for each day according to the Bayesian logistic regression estimates.θ the observed mean parameter computed from the original dataset.We then computed the natural history parameters for the 100 replicates and compared their distributions to the parameters computed directly from the observations. To quantify the accuracy of our predictions, we computed the relative bias as S1 Table(DOCX)Click here for additional data file.S2 Tablerse stands for relative standard error. The fixed effect represent the average population parameter value and the random effect represents the between pig variability.(DOCX)Click here for additional data file.S1 FigLeft panels: posterior distribution. Right panels: convergence traces.(TIFF)Click here for additional data file.S2 FigObservations are shown by the grey dots and best fit curves by the black lines. Each box represents a donor pig.(TIFF)Click here for additional data file.S1 File(ZIP)Click here for additional data file.S2 File(ZIP)Click here for additional data file.


R0) and effective (Re) reproduction numbers demonstrate that the ASF epidemic is declining and under control with an R0 of 1.139  = 1.123–1.153) and Re of 0.802 (95% CI = 0.612–0.992). In the last phases of an epidemic, these estimates are crucial tools for identifying the intensity of interventions required to definitively eradicate the disease. This approach is useful to understand if and when the detection of residual seropositive WB is no longer associated with any further ASFV circulation.African swine fever (ASF) severely threatens the swine industry worldwide, given its spread and the absence of an available licensed vaccine, and has caused severe economic losses. Its persistence in wild boar (WB), longer than in domestic pig farms, and the knowledge gaps in ASF epidemiology hinder ASF virus (ASFV) eradication. Even in areas where disease is effectively controlled and ASFV is no longer detected, declaring eradication is difficult as seropositive WBs may still be detected. The aim of this work was to estimate the main ASF epidemiological parameters specific for the north of Sardinia, Italy. The estimated basic ( Sus scrofa domesticus) and wild boar in Europe, and Phacoerus africanus or Potamochoerus spp. in Africa, makes its control and eradication even more difficult  is the prior distribution of eR, the denominator is a normalization factor, and the notification of new cases in the next reports allows the estimation of the probability distribution function of eR as the posterior [t  as the prior for new cases at t + τ. Uncertainty bounds  were obtained by extracting the average and maximum-likelihood values of eR by its estimated distribution.osterior ,55,56. TMATLAB software . The SEIR models based on the different R0 parametrizations were stochastically implemented in the software package Berkeley Madonna . The Bayesian stochastic SEIR model was implemented using R software ‘BRugs’  and Open BUGS (version 3.2.3).All parameter computations were solved using λ) estimated by the average contact rate was 0.0056 . As illustrated in td) of 39 days. Using this doubling time, we estimated R0 to be 1.124 (95% CI = 1.103–1.145) during the first two hunting seasons. As reported in R0 calculated based on λ was described by a value of 1.165 (95% CI = 1.027–1.187). Based on the proportion of infected, an R0 of 1.170 (95% CI = 1.009–1.332) was estimated for the first two years under study. The estimates of the reproduction number obtained by the three methods were found to be consistent with each other . The estimation method applied to the eR calculation for the last three years (2017–2020) showed a value of 0.802 (95% CI = 0.612–0.983).Based on the serological data of the first two hunting seasons described in R0 ASF-specific for the WB-MU of Anglona-Gallura, four different curves describing the total number of infected animals were estimated by the optimization method with the SEIR models (R0 of 1.124 described an infectious curve with an expected peak of 53 virus-infected wild boar at time point 729 (20 January 2014) and estimated the last peak at time 1315 (29 August 2015) (green line). The second SEIR fitted by the application of the λ value in the R0 calculation (R0 = 1.165) estimated a peak of 88 virus-positives at time 619 (2 October 2013) and the last at time point 1112 (7 February 2015) (blue line). The third system of differential equations implemented with an R0 of 1.170, obtained by the number of susceptible animals, predicted a total of 93 infected animals at time 610 (23 September 2013) and the last virus-positive individual at time 1091 .Considering the total wild boar population  at the start simulation point (22 January 2012), based on the different parameterizations of R models . The firR0 obtained using the SEIR optimized method was ≈ 1.139 (95% CI = 1.123–1.153), with a peak at time point 687 (9 December 2013) with 64 infected animals, and the last positive animal detected at time point 1236 (11 June 2015) (black solid and dashed lines). Considering the scatter plot and log-likelihood deviation, the values of R0 estimated by the third method  seems to more accurately describe the observed data with an overall deviance of 132 (10 degrees of freedom). The Bayesian SEIR model estimated the overall average of the effective reproduction number as 0.923 (BCI = 0.812–1.033) with level of uncertainty between observed and estimated (maximum likelihood) measured by the width of the BCI, which decreased as case observations increased  as a result of averaging periods in which the epidemic declined.A total of 164 cases  occurred in Anglona-Gallura with an epidemic peak date at time point 657 (9 November 2013), and the last infected animal detection occurred at time 1434 (25 December 2015). The best fitting value of ncreased . At lateR0 at first virus detection (PCR-positive) in wild boar ranged from 1.24 to 1.70, whereas the eR estimation of 0.802 suggests that the disease is now under control.Despite its importance, to date, few epidemiological methods have been proposed to determine the end of an epidemic and the ASF-free status of a territory ,20,57,58λ and R0 values proposed in the literature for ASF vary widely and should be considered as context specific.Even though the estimation of the main epidemiological parameters and particularly the effective reproduction number is fundamental to understanding the current status of an epidemic, studies focused on this aim are lacking in the literature ,28,29,59Several gaps in the ASF epidemiology still remain, generating additional uncertainty when defining the fade out of a virus in the wild boar population; the undefined role of surviving seropositive animals and their possible role as carriers are two of the most influential factors ,61,62,63λ parameters could be slightly underestimated, particularly those proposed by Methods 2 and 3. However, the large amount of data collected at least partially limited these biases.Although hunting is equally managed inside the IZ, as described by Cappai et al. , not allR0 and Re revealed that at the end of the PCR-positive detection period, eR progressed to and remained below zero and the infection faded out spontaneously despite the presence of seropositive wild boars. However, even if the mathematical values of the epidemiological parameters indicate that the virus is fading out, the conclusions have to be further confirmed:(1)In any European area, the sampling intensity achievable through recreational hunting will always be too low to detect the virus at a very low prevalence in localized pockets of infection ,66;(2)The efficiency of passive surveillance proves that the absence of the virus  is far from being known and standardized;(3)The absence of virus-positive animals among the hunted wild boar for a long time, as associated with serological data, provides valuable information for confirming that the disease is moving toward self-extinction; and(4)The seropositive and virus-negative animals that are still considered carriers despite their presence do not lead to any further virus detection, as confirmed by this study and in other similar epidemiological situation . Notably, ASFV-specific antibodies in a wild boar population were found several years after the fading out of the virus ,61,62,63Our findings confirmed that among the different ASF cycles , in SardIn this epidemiological landscape, hopefully observed for Euro-Asiatic ASF in the near future, it is necessary to further study and identify a specific technical approach that is able to merge epidemiological analyses, field data, and scientific evidence to minimize the risk of missing possible persistence of ASF at a very low prevalence and to ensure that restrictions are not maintained for too long, given low numbers of detected seropositive wild boars.


The twists to convert a cube to a square antiprism and a hexagonal prism to a hexagonal antiprism eliminate the need for the highest nodality orbitals in the resulting hybrids. A trigonal twist of an hO octahedron into a Dh3 trigonal prism can involve a gradual change of the pair of d orbitals in the corresponding sp3d2 hybrids. A similar trigonal twist of an hO cuboctahedron into a Dh3 anticuboctahedron can likewise involve a gradual change in the three f orbitals in the corresponding sp3d5f3 hybrids.The combination of atomic orbitals to form hybrid orbitals of special symmetries can be related to the individual orbital polynomials. Using this approach, 8-orbital cubic hybridization can be shown to be sp Such studies included an investigation of distortions from ideal symmetries in relatively symmetrical systems with molecular orbital degeneracies . Th. Th13]. 3, sp3d5, and sp3d5f7 manifolds in hybridization schemes should lead to most nearly spherical polyhedra , synthesized by Ghosh and co-workers [Dh3 tricapped trigonal prism found experimentally in the MH92−  anions [Fully using the complete spolyhedra . For thevertices . The tet-workers . There i) anions .8 metals such as Ni(II), Pd(II), Pt(II), Rh(I), and Ir(I). Since a square is a two-dimensional structure in an xy plane, the polynomials of the atomic orbitals for square hybridization cannot contain a z variable. This limits the p orbital involvement in square hybridization to the p{x} and p{y} orbitals so that a d orbital is required for square hybridization. This d orbital can be either the d{x2 − y2} or d{xy} orbital, depending on whether the x and y axes are chosen to go through the vertices or edge midpoints, respectively, of the square (pd2f) = sp3d3f set for cubic hybridization. This shows that one f orbital, namely the A2u{xyz} orbital with a cube orbital graph } set. Thus, the set of 10 orbitals for the pentagonal prism are sp3d4{x2 − y2,xy,xz,yz} f{xyz,z(x2 − y2)} without involvement of the d{z2} orbital. Capping the pentagonal prism in a way to preserve the Dh5 symmetry adds the d{z2} and f{z3} orbitals to the hybrid leading to an sp3d5f3{z3,xyz,z(x2 − y2)} set for the resulting 12-vertex bicapped pentagonal prism.The 10-vertex pentagonal prism of symmetry  is encou  and f(z3) orbitals to give an sp3d5f3 12-orbital hybridization scheme. This process is exactly analogous to the conversion of the square antiprism to the bicapped square antiprism discussed above.Twisting one pentagonal face of a pentagonal prism 36° around the symmetry . Unlike ndD symmetry of an n-gonal antiprism is preserved by compression or elongation along the major nC axis to give oblate or prolate polyhedra, respective, as illustrated in 5dD symmetry of the bicapped pentagonal antiprism to give the full icosahedral point group hI. This ascent in symmetry combines some of the irreducible representations of the Dh5 point group to give irreducible representations of higher degeneracy. As a result, the A1g + E1g + E2g irreducible representations coalesce into the single five-fold degenerate Hg representation of the icosahedral point group. The five d orbitals belong to this Hg representation in icosahedral symmetry. Similarly, the three f orbitals of the sp3d5f3 icosahedral hybridization belong to the single T2u{x3,y3,z3} irreducible representation of the hI point group considering the cubic set of f orbitals } or the f{y(3x2 − y2)} orbital depending on the {x,y,z} coordinate system chosen.The simplest structure with hexagonal symmetry is the planar hexagon itself. The restriction of the atomic orbitals to those containing no 3d3f set of hybrid orbitals for the hexagonal bipyramid is obtained by supplementing the sp2{x,y}d2{x2 − y2,xy}f{x(x2 − 3y2)} set of orbitals for the equatorial planar hexagon with the p{z} and d{z2} pair for the two additional axial vertices. The hexagonal bipyramid shares with the cube (see above) the feature of not arising from any sp3d4 hybridization scheme but instead requiring an f orbital in a sp3d3f hybridization scheme.The smallest three-dimensional figure with hexagonal symmetry is the 8-vertex hexagonal bipyramid . The sp3x and y in their orbital polynomials with an equal number of orbitals in which the polynomials obtained by multiplying the orbital polynomials for the planar face by the z variable. Thus, the sp2{x,y}d2{x2 − y2,xy}f{x(x2 − 3y2)} orbitals corresponding to the hexagonal face of the hexagonal prism is supplemented by the p{z3}d2{xz,yz}f2{xyz,z(x2 − y2)}g{xz(x2 − 3y2)} set of six orbitals. This leads to the interesting observation that a single g orbital corresponding to the B2g irreducible representation is required to provide a set of 12 hybrid orbitals oriented towards the vertices of a Dh6 hexagonal prism } for the resulting 12-vertex bicapped pentagonal prism.The atomic orbitals required to form a prism combine those for the polygonal face having only al prism . Not surC6 axis leads to the hexagonal antiprism of Dd6 symmetry  to the corresponding antiprisms involves a twist of (180/n)° of an n-fold face around the unique n-fold axis. Analogous processes can be applied to the three-fold axes in the regular octahedron and icosahedron. Such a twist process converts the hO regular octahedron into the D3h trigonal prism and the hO cuboctahedron into the Dh3 anticuboctahedron ,y(z2 − x2),z(x2 − y2)} set of the cubic f orbitals } or the Eu{x(3x2 − y2),y(3x2 − y2)} set. Thus, for the cuboctahedron, like the octahedron under 3dD symmetry, there are two alternative pairs of the required highest l value orbitals in the minimum l value hybrid that can be used in the hybrid. Converting the 3dD cuboctahedron to the 3hD anticuboctahedron eliminates the Eu{x(3x2 − y2),y(3x2 − y2)} pair of f orbitals from the sp3d5f3 hybrid requiring use of the E´{2,yzxz2} pair.The sporbitals . Reducinx and y. Since there are only two orbitals for each non-zero l value and one orbital  for l = 0, square planar hybridization requires one d orbital, pentagonal planar hybridization requires two d orbitals, and hexagonal planar hybridization requires an f orbital as well as two d orbitals. Prismatic configurations with two parallel n-gonal faces and nhD symmetry combine the set of atomic orbitals for the n-gonal face with only the two x and y variables with a set of equal size corresponding to the orbitals having polynomials in which the polynomials of the planar n-gon orbitals are multiplied by a third z variable. As a result, cubic hybridization can be shown to require an f orbital and hexagonal prismatic hybridization can be shown to require a g orbital. For 8-coordinate systems with four-fold symmetry twisting a square face of a cube with sp3d3f hybridization by 45° around a C4 axis to give a square antiprism with sp3d4 hybridization eliminates the need for an f orbital in the resulting sp3d4 hybridization. Similarly, for 12-coordinate systems with six-fold symmetry, twisting a hexagonal face of a hexagonal prism with sp3d5f2g hybridization to a hexagonal antiprism eliminates the need for a g orbital in the resulting sp3d5f3 hybridization. A trigonal twist of an hO octahedron into a Dh3 trigonal prism can involve a gradual change of the pair of d orbitals in the sp3d2 hybrids. A similar trigonal twist of an hO cuboctahedron into a Dh3 cuboctahedron can likewise involve a gradual change in the three f orbitals in the sp3d5f3 hybridization.The combination of atomic orbitals to form hybrid orbitals of special symmetries can be related to the individual orbital polynomials. Thus, planar hybridizations such as the square, pentagon, and hexagon can only use atomic orbitals with only two variables in their polynomials, conventionally designated as