Aerosols or saliva containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can contaminate living environments, and viruses can be indirectly transmitted. To understand the survival potential of the virus, the viral titers of bovine coronavirus (BCoV), as a model virus, and SARS-CoV-2 were measured on porous and non-porous surfaces. The amount of infectious BCoV recovered remained relatively high on non-porous substrates. However, it quickly decreased on several non-porous surfaces such as nitrile rubber. The time taken to reach the limit of detection on non-woven masks, as a porous substrate, was longer than that of non-porous substrates. On porous substrates other than non-woven masks, the amount of virus recovered quickly decreased, and then remained at a low level. Representative substrates were tested with SARS-CoV-2. The decrease in the amount of infectious virus recovered was similar to that of BCoV, although that of SARS-CoV-2 was more rapid. RNA derived from SARS-CoV-2 was also detected using real-time PCR, and it remained on surfaces much longer than infectious virus, on all substrates. Therefore, it is important to measure the viral titer to avoid the overestimation of infectious virus contamination in the environments. Our results suggest that the surface structure was not directly related to viral survivability. In the case of COVID-19, surveys of hospitals and cruise ship environments have revealed the presence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) on various surfaces with which patients came into contact, such as door knobs, TV remote controls, phones, floors, and bedding3. SARS-CoV-2 particles on the surfaces of various materials may therefore be a source of infection, a hypothesis which has been supported by several epidemiological studies5. The amount of virus on a surface and the length of time for which viruses survive on materials are crucial for indirect transmission.With viral respiratory infections, transmission of the virus often occurs through direct transmission. Direct transmission requires respiratory droplets and aerosols generated by coughs, sneezes, and conversation. It is also believed that respiratory viruses can be indirectly transmitted10, with differing results. These differences may be attributable to differences in the experimental conditions. The relative humidity and temperature influence the evaporation of fluids containing virus particles. With respect to surface structure or roughness, previous researchers have used the terms “porous” or “non-porous” to refer to the surfaces of materials, indicating a rough and coarse, or smooth texture of the surface, respectively. Because porous materials, such as paper and non-woven masks, contain multiple grooves and spaces, they absorb fluid, and trap substances in the fluid. In contrast, non-porous material cannot absorb fluid. The surface properties of a material therefore greatly influence the amount of virus which can be recovered from a surface. Although previous studies used the same materials, including plastic and glass, the authors did not describe the details of the substrates, such as the kinds of plastics, metals, and wood, or the characteristics of the surfaces of these substrates. Unless studies use materials with similar characteristics, the results will vary. Previous studies have mainly investigated non-porous materials, and there are few data pertaining to porous materials. However, individuals constantly come into contact with porous materials such as masks, bank notes, wood, clothing, and paper in the course of daily life. Other factors may be involved in viral recovery from substrates, in addition to surface structure or roughness.Various factors, such as relative humidity, temperature, and the surface properties of a material, affect the amount of infectious virus which can be recovered from a surface. The recovery of infectious SARS-CoV-2 from the surfaces of various materials has been studied by several groupsIn previous studies, SARS-CoV-2 was detected either by infecting cultured cells or by demonstrating the presence of viral RNA. The infection of cultured cells directly demonstrates the presence of infectious virus, but the detection of RNA reflects the presence of both infectious and non-infectious virus. Therefore, there can be discrepancies between the recovery time of infectious virus as measured by infectivity and that measured by the presence of viral RNA.Betacoronavirus, as does SARS-CoV-2, reported by previous studies based on the molecular phylogenetic analysis that BCoV is one of the species most closely related to SARS-CoV-211. After classifying the surfaces into several categories, we investigated the survivability of SARS-CoV-2 on the surfaces of representative characterized materials.To address these problems, we prepared various substrates of known origin, and measured their surface roughness. We detected the virus using both infection of cultured cells and the presence of viral RNA. Experiments with SARS-CoV-2 should be performed in a biosafety level (BSL) 3 laboratory. To facilitate the investigation of a wide range of different surfaces, we initially used bovine coronavirus (BCoV) handled in a BSL2 laboratory as a substitute for SARS-CoV-2. This virus belongs to the genus The area surface roughness of each representative substrate was measured, and shown as 3D microscope images Fig. . The mic10 TCID50/mL. Because of the cytotoxicity of the highest dilution of virus recovered from liquid from brass, nitrile rubber, and lauan veneer, the LoD was determined to be 1.4 log10 TCID50/mL. Using the slopes of the line of best fit, as shown in Fig. 10 TCID50 as an indicator of viral infectivity, assuming a linear decrease in viral recovery based on similar studies12 for all substrates .The two viruses had the common characteristics; the virus recovery from only surfaces of copy paper was significantly reduced by about 4 log10 RNA copies/mL, and the remaining number of SARS-CoV-2 RNAs diminished over time on all five substrates. The virus copy number was greatly reduced from 8.2 to 5.6 log10 RNA copies/ml after 0 h on copy paper. The LoD in this RT-PCR assay was 4.8 log10 RNA copies/mL  are also used as fungicides19, and it has also been reported that nitrile rubber has biocidal activity20. We therefore evaluated the type of the trend of the recovery of infectious virus on brass, soft polyvinyl chloride and nitrile rubber as “rapidly decreasing”  took 21.5 h, as calculated from the slope of the line of best fit for the non-woven-mask in this study . Purified BCoV was diluted tenfold in DMEM-high glucose with the supplements described above, and used in inoculation experiments on surfaces.For the propagation of BCoV, HRT-18G cells were cultured in Dulbecco’s Modified Eagle’s Medium high glucose  supplemented with 10% FBS  and 1% Antibiotic–Antimycotic (Thermo Fisher Scientific)26. After 5 days at 37 °C in 5% CO2, cell supernatants and virus were collected, and diluted tenfold in DMEM-low glucose without supplements, and used in inoculation experiments on surfaces.For the propagation of SARS-CoV-2, VeroE6/TMPRSS2 (JCRB 1819) cells were cultured in Dulbecco’s Modified Eagle’s Medium low glucose  supplemented with 10% FBS, 2.5 µg/mL G418 disulfate aqueous  and 1% Antibiotic-AntimycoticThe surfaces of 15 commercially available substrates representing a variety of surfaces in living environments were tested in this study (Supplementary Table n = 8).The area surface roughness (µm), which expresses, as an absolute value, the difference in height of each point compared to the arithmetical mean of the surface, was measured using a VK-X1000 3D microscope by Keyenece . The imaging field was 705 µm in the X axis and 528 µm in the Y axis (× 20 magnification). Sa was calculated as the average value of the measurements taken at eight non-overlapping observation surfaces on the sample surface , and after 3 h, 6 h, and 18 h, in 300 µL drops of DMEM-high glucose supplemented with 1% FBS, 1% Antibiotic–Antimycotic and 2.5 µg/ml pancreatin  by pipetting on the surface for a minute, and collecting as much as possible of the fluid into a microtube. SARS-CoV-2 was recovered at five time-points; time 0 h (immediately after drying), and after 3 h, 6 h, 12 h, and 24 h, in 300 µL drops of DMEM-low glucose supplemented with 1% FBS, 2.5 µg/ml G418 and 1% Antibiotic–Antimycotic by pipetting on the surface for a minute, and collecting as much as possible of the fluid into a microtube. Each recovery of virus was repeated twice on each surface, and a total of 600 µl of inoculum was collected. The negative controls consisted of the same surfaces without prior inoculation of the virus. The infectious virus titer in a recovery collected at each time point was compared, to evaluate the efficiency of infectious virus recovery. This experiment was repeated in triplicate.After drying, the substrate was placed in a glass Petri dish in a constant temperature and humidity incubator set at 75% RH and 25 °C 2. BCoV was inoculated onto fresh monolayers of HRT-18G cells in a 96-well plate. The culture was maintained for 6 days at 37˚C in 5% CO2. After incubation, the cells were monitored to detect virus-induced cytopathic effect (CPE). The virus titers were calculated using the Reed–Muench method30.The amounts of infectious virus on all surfaces were quantified using end-point titration with a tenfold serial dilution of virus recovery in DMEM with the supplements described above. SARS-CoV-2 was inoculated onto fresh monolayers of cells (Vero E6/TMPRSS2) in a 96 well plate. The culture was maintained for 4 days at 37˚C in 5% CO10 TCID50/mL). The cytotoxicity was observed at the highest dilution of a virus recovering liquid from three kinds of substrates, such as brass, nitrile rubber, and lauan veneer. Using this estimation, the LoD in the viral titer assay was determined as 0.6 log10 TCID50/mL for SARS-CoV-2, 0.4 log10 TCID50/mL for BCoV with non-cytotoxic materials, and 1.4 log10 TCID50/mL for BCoV with cytotoxic substrates, as described above. An assay without CPE was regarded as ND. For the calculation of the mean values and standard deviations of titers, ND was regarded as 0 TCID50/mL. The titer declining lines were calculated by linear regression using the mean titers, excluding all ND data in a time point. For the comparison of decreasing rates, two-sided paired-samples t-test was performed with the significance level at p < 0.05.When the titers were below the limit of quantification of the Reed–Muench method, the titer value was estimated by dividing the numbers of wells in which CPE appeared by the equivalent volumes of undiluted recovering liquid in an assay . To quantify the viral RNA copy numbers, a standard curve was generated using Positive Control RNA Mix . The time to LoD for SARS-CoV-2 was calculated at a detection limit as 4.8 log10 RNA copies/mL for all substrates.Viral RNAs were extracted from the SARS-CoV-2 recoveries from surfaces using the QIAamp Viral RNA Mini Kits  following the manufacturer’s instructions. RT-PCR was performed using One Step PrimeScript RT-PCR Kits with Primer/Probe set N2 Supplementary Information.


Cryptococcus gattii, which has allowed for the phylogenomic specification of previously described major molecular type groupings and novel lineages. Recently, isolates collected in Mexico in the 1960s were determined to be genetically distant from other known molecular types and were classified as VGVI. We sequenced four clinical isolates and one veterinary isolate collected in the southwestern United States and Argentina from 2012 to 2021. Phylogenomic analysis groups these genomes with those of the Mexican VGVI isolates, expanding VGVI into a clade and establishing this molecular type as a clinically important population. These findings also potentially expand the known Cryptococcus ecological range with a previously unrecognized endemic area.Whole-genome sequencing has advanced our understanding of the population structure of the pathogenic species complex  Cryptococcus species complexes (C. gattii and C. neoformans). However, genomic tracking of Cryptococcus is seldom done for the estimated 223,100 annual clinical meningitis cases worldwide [Cryptococcus subpopulations into molecular types and proposed them as separate species [C. gattii populations that cause illness have been characterized and sequenced, and comprise mostly the molecular types VGI, and VGII, although the VGIII [C. gattii molecular type VGV, which was found via environmental sampling in central Zambian woodlands [C. decagattii [Whole-genome sequencing has provided an important element in defining the population structure for many human fungal pathogens, including the he VGIII  and VGIVhe VGIII  genomic oodlands , as welloodlands  and an ioodlands . These cC. gattii collected from 2012 to 2021 in the southwestern United States and Argentina. Here, we present the genomes of these isolates and their phylogenomic placement in the species tree.We sequenced four clinical isolates and one veterinary isolate of C. gattii that were sequenced in this study, three were collected from patients with pulmonary or meningeal cryptococcosis in Arizona and classified as C. gattii by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) (Of the five isolates of try (MS) . The geotry (MS) .C. gattii species complex, including the “VGVI” genomes that originated from Mexico in the 1960s and 1987 . Read files for each sample were considered typed when read counts assigned to the top match in the custom database surpassed those of the second-best match by a factor greater than two. Draft genomes were assembled for use as references for single nucleotide polymorphism (SNP) detection, using SPAdes v3.10.1 with the “careful” flag activated [Genome libraries were prepared with a DNA prep kit (Illumina) at one-fourth of the reaction volumes and sequenced on a NextSeq 550 (Illumina). Molecular typing was performed over the sequence reads via a custom 31-mer Kraken2 database  consistictivated .Subsequently, sequence reads were compared to public genomes, including that of their putative molecular type VGVI  by SNP-based phylogenetic inference. Reads were mapped to reference genomes using BWA v0.7.17  and SNPsC. gattii molecular types —illu pockets ,30, and  pockets ,32. Of nC. gattii VGVI is a possible risk for patients in previously undescribed endemic regions of the globe. A lack of clear exposure history for the Arizona human patients prevented confirmation of endemic locales at this time. Nevertheless, the link to the southwestern United States was compelling when the provenance of the isolates was considered; for example, clinical isolate 7685027, collected in southern California, and reported by Springer et al. [C. gattii in Arizona was previously published [C. gattii.The basal placement of the Argentinian isolate in the VGVI phylogeny shows the immediate relation of the isolate to the North American cases and indicates that r et al. , likely r et al. . The locThe VGVI population structure showed substantial differences across samples B, which Cryptococcus molecular typing is rare in clinical settings, but new technologies now make it a possibility. The use of MALDI-TOF MS in the Arizona clinical microbiology laboratory in this study is a recent change. Previous methods may have misclassified C. gattii infections as C. neoformans, which is the more likely species to be encountered in this setting. The use of MALDI-TOF MS for rapid, simple, and reliable molecular type identification in Cryptococcus spp. was demonstrated in research applications [ications ,34,35; MCandida auris [C. gattii VGII [C. gattii VGVI. Further surveillance that includes typing methods, especially whole-genome sequencing, will provide the opportunity to clarify the endemicity of VGVI and to ascertain whether this pathogen represents an emerging risk to susceptible human and animal hosts in the identified regions.Tracking the geographical origin and the dynamics of spread is especially challenging in fungi, due to their complex ecology and the large timescales, usually millennia, that encompass the evolution of these organisms ,36. Recoda auris ,38 and Ctii VGII . These a


Given the rising incidence of young-onset colorectal cancer (yCRC) among individuals younger than 50 years old, understanding the economic burden of yCRC is required to inform the delivery of healthcare services. Therefore, we conducted a systematic review of studies assessing the direct medical costs of yCRC, and where relevant average-age onset CRC (aCRC).We searched MEDLINE, EMBASE, and Web of Science from inception to May 2022 for original, peer-reviewed studies, that reported direct medical costs  for yCRC and aCRC. We used a modified version of the Consolidated Health Economic Evaluation Reporting Standards checklist to appraise the studies. Costs were inflation-adjusted to 2020 US dollars.We included 14 studies from 10 countries, including the USA, England, France, Korea, Vietnam, China, Italy, Australia, Canada and Japan. Five studies focused on prevalent disease and reported annualized per-capita cost of prevalent yCRC, ranging from $2,263 to $16,801 and $1,412 to $14,997 among yCRC and aCRC cases, respectively. Nine studies estimated the cost of incident disease. Synthesis of per-capita costs incurred 12 months following colorectal cancer diagnosis ranged from $23,368 to $89,945 for yCRC and $19,929 to $67,195 for aCRC. Five studies used multivariable approaches to compare costs associated with yCRC and aCRC, four showed no differences and one suggested greater costs with yCRC.Our synthesis of direct medical costs of yCRC across multiple jurisdictions provide relevant information for healthcare decisions, including on-going considerations for expanding CRC screening strategies to younger adults.The online version contains supplementary material available at 10.1186/s12913-022-08481-6. Colorectal cancer (CRC) is the third most common malignancy and the second most deadly cancer worldwide . With reWith the increasing risk of yCRC, comes the need for understanding its economic impact; of particular interest are direct medical costs – costs that are directly related to provision of medical services for patient care . Additionally, individuals with yCRC are more often diagnosed at later stages and with metastatic disease in comparison to individuals with aCRC, largely due to the lack of age-specific diagnostic guidelines . As a reWe conducted a literature search of Ovid MEDLINE, Ovid Embase and Science Citation Index and Social Sciences Citation Index via Web of Science from inception to July 15, 2021, and then updated the search on May 30, 2022. To ensure comprehensive capture of articles that may assess yCRC as a subgroup of CRC, we employed a broad search strategy to identify articles on CRC across all ages, from which data pertaining to yCRC could be extracted. Our search strategies used a combination of subject headings  and keywords to locate studies. Search terms related to economics/costs were adapted from a search filter developed by Canadian Agency for Drugs and Technologies in Health  (Tables We reviewed titles and abstracts to identify published studies that met our systematic review inclusion criteria of: 1) an original study; 2) published in a peer-reviewed journal as a full-length article; 3) patient population with CRC or yCRC; 4) published in English; and 5) reported the direct medical cost of yCRC, defined as costs directly related to provision of medical services for patient care . Given potentially different age cut-offs for defining ‘young-onset’, for the purposes of our systematic review, we broadly considered an age cut-off of up to 65 years old in their cost estimation and reported as yCRC. We excluded non-original literature , economic evaluations , studies which estimated costs associated with co-morbidities related to CRC or side effects of cancer treatment, studies referring to the cost of CRC screening, studies comparing costs associated with different cancer treatment protocols, and conference proceedings. While the aim of our study was to extract the direct medical costs associated with CRC treatment, we also accepted studies that included cost of illness prior to diagnosis in their definition for CRC treatment, as many countries lack standard screening protocols, resulting in increased spending prior to pathological confirmation of diagnosis. Three authors  independently reviewed the titles and abstracts of articles identified from literature search and resolved discrepancies by consensus. Abstracts that met our inclusion criteria were forwarded for full-text review. The same three authors independently assessed articles eligible for a full-text review based on the inclusion criteria. To assess included studies for quality, we used a modified version of the Consolidated Health Economic Evaluation Reporting Standards (CHEERS) checklist , which iTo characterize the included studies, we extracted information on country, data source, length of follow-up, cancer site , sex, sample size, and age-cut off used for estimating and reporting costs. Of particular interest in our systematic review, we extracted detailed information on: 1) costing approach, such as source of payment ; 2) payer perspective ; 3) whether costs were attributable to  or associated  with CRC; and 4) cost components . We then extracted the reported per-capita direct medical costs for both prevalent  and incident cases of yCRC, and where relevant, aCRC. Also, where relevant, we extracted information on cost comparisons between yCRC and aCRC cases, specifically those based on the use of multivariable approaches. For brevity in our reporting of results, we use the term ‘costs’ to refer to direct medical costs. To facilitate comparisons, all extracted costs were inflation-adjusted to 2020 USA dollars (USD) using the Consumer Price Index unless otherwise specified. Costs in the original currency are provided in Table SOur search strategy resulted in 17,764 articles on July 15, 2021 and 1,584 articles in the May 30, 2022 update  [n = 9) [Characteristics of studies included in the systematic review are shown in Table n = 5) –14 or in [n = 9) –23. Stud [n = 9) , 17, 19, [n = 9) , 18, Fra [n = 9) , China [ [n = 9) , 20, Kor [n = 9) , Vietnam [n = 9) , Italy [ [n = 9) , Canada  [n = 9)  and Japa [n = 9) , 13, 20, [n = 9) , 18, 22, [n = 9) , lastly  [n = 9) . It must [n = 9) , 21 or h [n = 9) , 13, 20, [n = 9) , 20, and [n = 9) , 12. Wit [n = 9) , 11, one [n = 9) , 18–23 s [n = 9) , 17 inclTable p = 0.419), 45–54 (p = 0.131) and 55–64 (p = 0.522) years old compared to those diagnosed at 65 (reference) years or older [Table or older . Among p54 p = 0.1 and 55–Among more recent studies, Gigli et al., used administrative health data to estimate costs directly related to rectal cancer treatment  by phases of care . Specifin = 5) of incidence-based costing studies used an age cut-off of less than 65 years old [Nine studies estimated the per-capita cost of incident CRC cases, with differences between the selected time horizon, point of care  and cancer site . Of note, in contrast to prevalence-based costing studies that reported age cut-offs consistent with current definitions of yCRC , the majority  . Here, aUtsumi et al., used national health insurance claims data in Japan and estimated costs associated with CRC stratified by age and treatment type . SpecifiLastly, two out of the nine included studies stratified cost estimates by age and cancer site , 21. TapIn light of the increasing risk of yCRC , 24, 25,To our knowledge, direct medical costs associated with yCRC have not been systematically evaluated. While Yarboff et al., conducted a systematic review of studies that estimated the economic burden of CRC in 2013, the authors largely focused on evaluating costing methodologies of the included studies . SpecifiAside from synthesizing reported costs, observed trends across included studies have implications for better understanding of yCRC. For example, as demonstrated by findings from Ritzwoller et al., and Laudicella et al., costs associated with late stage (stage III/IV) or metastatic CRC were particularly pronounced among yCRC patients, with Rizwoller et al., reporting the cost associated with a metastatic cancer diagnosis to be $89,945 among yCRC patients, compared to $67,195 among aCRC , 19. AddWhile the aim of our study was to capture costs for yCRC, which has largely been defined among adults diagnosed with CRC before the age of 50 years , due to The strengths and limitations of our systematic review warrant discussion. To ensure a thorough literature search, we developed our search strategy in collaboration with an information scientist who executed all database searches. An original and updated search further ensures comprehensive and timely capture of relevant studies to date. While we standardized all costs to 2020 USD to facilitate interpretation, we caution comparison of costs estimates across different jurisdictions due to differences in the delivery and cost of healthcare services, which also prohibited us from pooling the data . As the focus of our study was on direct medical costs of yCRC, we did not synthesize indirect costs such as productivity loss, which is relevant given that as young adults, individuals with yCRC comprise a greater majority of the work-force. As mentioned earlier, for the purposes of our systematic review we considered individuals diagnosed with CRC younger than 65 years old as ‘young-onset’. As such our synthesis may not accurately represent our target population of younger adults diagnosed with CRC.In conclusion, synthesis of available evidence suggests that the per-capita costs of yCRC is substantial and does not significantly differ from the per-capita costs of aCRC. Given the global rise in the incidence of yCRC  and evidAdditional file 1:Supplementary Table-S1. Database(s): Ovid MEDLINE(R) and Epub Ahead of Print, In-Process & Other Non-Indexed Citations, Daily and Versions(R) 1946 to July 13, 2021. Supplementary Table-S2. Database(s): Embase 1974 to July 14, 2021. Supplementary Table-S3. Database(s): Web of Science, Science Citation Index and Social Science Citation Index only. Supplementary Table-S4. Quality assessment of included studies. Supplementary Table-S5. Direct medical costs associated of yCRC versus aCRC, original currency and inflation adjusted to 2020 USD.


Chromosomes in the eukaryotic nucleus are highly compacted. However, for many functional processes, including transcription initiation, the 3D pair-wise motion of distal chromosomal elements, such as enhancers and promoters, is essential and necessitates dynamic fluidity. Therefore, the interplay of chromosome organization and dynamics is crucial for gene regulation. Here, we use a live imaging assay to simultaneously measure the positions of pairs of enhancers and promoters and their transcriptional output in the developing fly embryo while systematically varying the genomic separation between these two DNA loci. Our analysis reveals a combination of a compact globular organization and fast subdiffusive dynamics. These combined features cause an anomalous scaling of polymer relaxation times with genomic separation and lead to long-ranged correlations compared to existing polymer models. This scaling implies that encounter times of DNA loci are much less dependent on genomic separation than predicted by existing polymer models, with potentially significant consequences for eukaryotic gene expression. Living systems are built based on information encoded in chromosomes confined in each cell’s nucleus. These meter-long DNA polymers must be highly compacted to fit into the micron-sized structure , 2. At tOver the past decade, the advent of chromosome capture and imaging methods  has giveSimilarly, from a polymer physics perspective, there is a gap in our understanding of static and dynamic properties of chromosomes. At large scales, across tens to hundreds of TADs, chromosome organization has been suggested to be highly compacted in a crumpled chain configuration , a long-lived polymer state with fractal dimension three , 29–32. in vivo.Thus far, experimental data sets have given insight into either static organization , 22, 32,Here, we address this problem by live imaging the joint dynamics of two cis-regulatory DNA elements, an enhancer, and a promoter, while simultaneously monitoring the transcriptional output resulting from their functional dynamic encounters in developing fly embryos. We systematically vary the genomic separation between these loci spanning many TADs. Stochastic real-time trajectories of the 3D motion of the two loci show a dynamic search process, with physical proximity required for successful transcription and a power-law scaling of transcription probability with genomic separation. While typical 3D distances between the locus pair follow a compact packing consistent with the crumpled chain model, the dynamic properties exhibit fast diffusion, albeit with a diffusion coefficient that increases with genomic separation. These features give rise to an anomalous scaling of polymer relaxation times and long-range correlations in the relative motion of the two loci. This suggests that the enhancer–promoter search process is much less dependent on genomic separation than expected based on existing polymer models.Drosophila even-skipped (eve) locus  locus . The locg system  8]. To . To Drosve locus .s varying over close to two orders of magnitude from 58kb to 3.3Mb, comparable to the distances over which many enhancers function in higher eukaryotes .We build seven of such reporter constructs, with genomic separations karyotes  9–12]. –12. s vaof 90 kb   of embryo development , well af<10kb . Therefooff where the homie elements are not bound to each other, and two paired configurations Poff and Pon, where a loop is formed with either inactive or active transcription, respectively. To determine the instantaneous topological and transcriptional state of the system, we adopt an inference approach using a Hidden Markov Model based on the time series of inter-locus distance and transcriptional activity  is likely dominated by the search of the two loci to come into contact. Importantly, different polymer models make distinct predictions of the scaling of contact probabilities [In contrast, the overall probability of observing either of the paired configurations strongly decreases with genomic separation, and exhibits a power-law scaling =0.9±0.2 . Since tbilities , 31, 51:t f≈1.15 , which ihomie insulator-mediated focal contacts in our system, we employed a reporter construct in which the homie sequence is replaced by a λ DNA sequence of the same length. Interestingly, at 58 kb separation transcriptional encounters still occur, albeit with a shorter median lifetime of  is the 3D position of the locus, Γ the diffusivity, and β the dynamic exponent. This exponent quantifies how locus diffusion scales with time and can be related theoretically to the packing of the chromosome via the fractal dimension eve locus (blue) and for the ectopic reporter (green), which is close to the prediction of the ideal chain model, and consistent with previous works [We quantify how this search process is reflected in the motion of individual DNA loci by computing the single-locus MSD =2/(2+d) , 39, 53.) [β=1/2 , we expe polymer . Our sysus works , 27, 35 us works , as theyR(t), we compute the two-locus MSD ??a,b). Consistent with the observed single-locus dynamics, we find that also the small-time limit of the experimental two-locus MSDs exhibits an exponent close to the ideal chain with 1/2 . Similarly, for large time-lags, the two-locus auto-correlation reveals agreement with the ideal chain scaling . Thus, the full time-dependence of the MSD is well described by the ideal chain predictions, both for single and coupled loci.To further understand how the locus dynamics are determined by the interplay of chromosome organization and single-locus dynamics, we analyze the joint dynamics of the two coupled chromosomal loci. From the statistics of the 3D distance vector t0))2〉t0 , which qτ, which corresponds to the time scale of the crossover of the two regimes of the two-locus MSD . Specifically, τ is the time taken by the two loci to diffuse (dynamics) over their typical distance of separation (organization): Having established the static and dynamic properties of the system, we now ask about the consequences of these features for the time scales of the two-locus search process. This process is determined by the interplay of chromosome dynamics and organization and can be characterized by a relaxation time ??e). This scaling appears to plateau for the largest genomic separation (3.3Mb) at a value close to the single locus diffusion, which remains approximately constant across separations . Notably, the absolute value of the diffusivity at the plateau is almost 20-fold larger than previous measurements in mammalian stem cells with similar genomic separation [To infer the relaxation time in our data as a function of genomic separation, we perform a Bayesian fitting of the two-locus MSD with the ideal chain expression  Supplem. Interesparation , suggest??f). This exponent corresponds to a much shallower scaling with separation than predicted by either the ideal or crumpled chain theory. This result is further confirmed by a data collapse of the two-locus auto-correlation functions  [τ, we can therefore make a parameter-free prediction of the correlations, which decay significantly more slowly than for the ideal Rouse model . Notably, we find that the experimental correlations are quantitatively captured by this parameter-free prediction , including the full time-dependence of the correlations  . Having elations . This dein vivo imaging of the joint dynamics of enhancer–promoter pairs with varying genomic separation and simultaneous monitoring of their transcriptional output. Observing the dynamics of pairs of DNA loci has only become possible recently and has been done for tagged DNA loci at a single fixed genomic separation [We developed an experimental approach to perform paration , 27, 36.Drosophila.Many features of the two-locus dynamics, including the subdiffusive exponent close to 0.5, are very well conserved with measurements of CTCF sites at TAD boundaries in mammalian systems , 27, desan cells , 27. Theoff state correlate well with the relaxation times, but are approximately 10 times larger on average  would be ~ 3000 times longer than for the shortest 58 kb separation. Our measurements however reveal that it only takes ~ 20 times longer, corresponding to a more than 100-fold reduction. This reduced dependence on distance implies that the probability of reaching and maintaining the spatial proximity required for transcription is similar for enhancers dispersed across the chromosome, allowing them to find their target promoter efficiently. This might be one of the reasons for why evolution can act on distal sequences from a given target promoter. Overall, our findings have crucial implications for the spatiotemporal organization of the cell nucleus, including the dynamics of long-range focal contacts  and mammFrom a polymer physics perspective, our measured exponents suggest that the relationship between static and dynamic properties in the generalized Rouse framework, which relies on the assumption of local friction, does not apply to chromosomes. This suggests that long-range interactions, such as hydrodynamics or active motor-mediated interactions , 57 coul-linking , out-of--linking , or the -linking –20. Toge-linking , 59–64 tSupplement 1


Here, we describe morphological and molecular alterations in gastric mucosa of slc26a9−/− mice and in selective parietal cell-deleted slc26a9fl/fl/Atp4b-Cre mice and correlate SLC26A9 expression levels with morphological and clinical parameters in a cohort of gastric cancer (GC) patients.Solute carrier family 26 member (SLC26A9) is a ClThe expression patterns of genes related to transport and enzymatic function, proliferation, apoptosis, inflammation, barrier integrity, metaplasia and neoplasia development were studied by immunohistochemistry (IHC), quantitative RT-PCR, in situ hybridization and RNA microarray analysis. SLC26A9 expression and cellular/clinical phenotypes were studied in primary human GC tissues and GC cell lines.−/HCO3− exchanger AE2, G2/M cell cycle arrest and apoptosis and suppressed their proliferation, migration and invasion.We found that both complete and parietal cell-selective Slc26a9 deletion in mice caused spontaneous development of gastric premalignant and malignant lesions. Dysregulated differentiation of gastric stem cells in an inflammatory environment, activated Wnt signaling, cellular hyperproliferation, apoptosis inhibition and metaplasia were observed. Analysis of human gastric precancerous and cancerous tissues revealed that SLC26A9 expression progressively decreased from atrophic gastritis to GC, and that downregulation of SLC26A9 was correlated with patient survival. Exogenous expression of SLC26A9 in GC cells induced upregulation of the ClOur data indicate that SLC26A9 deletion in parietal cells is sufficient to trigger gastric metaplasia and the development of neoplastic lesions. In addition, we found that SLC26A9 expression decreases during human gastric carcinogenesis, and that exogenous SLC26A9 expression in GC cells reduces their malignant behavior.The online version contains supplementary material available at 10.1007/s13402-022-00672-x. Helicobacter pylori (H. pylori) infection and genetic, epigenetic and environmental factors in a multistep process of histological progression from atrophic gastritis (AG) through intestinal metaplasia (IM) to GC [Gastric cancer (GC) is a common malignant tumor and a major health threat worldwide. Gastric carcinogenesis is believed to result from interactions between M) to GC , 2. In tM) to GC , 2.− uniporter that is strongly expressed in gastric surfaces and glandular epithelia in both mice and humans [Solute carrier family 26 member 9 (Slc26a9) is a member of the Slc26a family of multifunctional anion transporters  and has d humans –7. Previd humans . Parietad humans . The impFor detailed additional method descriptions see Supplementary Materials.Slc26a9fl/fl mice with a C57BL/6 J background were obtained from Cyagen Biosciences, China. Atp4b-Cre mice [Slc26a9fl/fl mice were crossed with Atp4b-Cre mice to produce parietal cell-specific Slc26a9 knockout Slc26a9fl/fl/Atp4b-Cre mice with a mixed genomic background. The mice were bred and genotyped in accordance with the Institutional Animal Care and Use Committee (IACUC) at Hannover Medical School, Germany, and at Zunyi Medical University, China, respectively. All mice ranging from 8 days to 18 months old were age- and sex-matched. All experiments involving animals were approved by the Hannover Medical School and Zunyi Medical University committees on investigations involving animals and an independent committee assembled by the local authorities.The Slc26a9-deleted mouse strain used, whose establishment and characteristics have been described elsewhere , 5, is cH. pylori negativity was confirmed by histologic examination for H. pylori and a C14 urea breath test), and chronic atrophic gastritis (CAG) samples were obtained from the Endoscopy Center and 145 GC tissues from the Department of Pathology of the Affiliated Hospital of Zunyi Medical University, from January 2010 to January 2019. The study was performed in accordance with the Second Helsinki Declaration and was approved by the Human Subject Committee of the Affiliated Hospital of Zunyi Medical University, Zunyi, China. All patients involved provided written informed consent.A tissue microarray containing 90 cases of multiple human GC tissues (HStm-Ade180Sur-06) was obtained from Shanghai Outdo Biotech. Human samples, including normal gastric epithelium . Survival curves were plotted using the Kaplan–Meier method and compared using log-rank tests. All analyses were performed at least in triplicate. The results are reported as the means ± SD. Significance was defined as *slc26a9−/− and wild-type littermates aged 8 days to 18 months using a murine gastric histopathology scoring system [1slc26a9−/− mice were not significantly different from those of wild-type mice (WM) at 8 days after birth. Parietal cell loss was observed at 1 month of age, oxyntic atrophy  with elongated and dilated glands was observed at 2 months of age and mucous cell metaplasia, including spasmolytic polypeptide-expressing metaplasia (SPEM) and intestinal metaplasia (IM), was observed at 6 months of age  and CHIA, respectively  and the chief cell marker Mist1  markers, including Lrig1, Lgr5 and Mist1, were downregulated in Slc26a9fl/fl/ATP4b-Cre mice as determined by transcriptome analysis , cleaved Caspase 9, cleaved Caspase 3, apoptosis-inducing factor (AIF) and endonuclease G (Endo G) were downregulated, indicative of the suppression of caspase-dependent and caspase-independent apoptosis  or below (SLC26A9 low) the median. By doing so, we found that the patients in the SLC26A9 low-expression group had a lower overall survival than those in the SLC26A9 high-expression group  in the CG cell lines as well as in SLC26A9-transfected AGS cells  and a resultant cellular Cl− overload. Since AE2 functions as a cellular acid loader, low AE2 activity will result in a high intracellular pH, as has been shown for intestinal crypt cells [i as a consequence of the restoration of a Cl− efflux pathway by Slc26a9 overexpression in AGS cells and a concomitant upregulation of an endogenous HCO3− export pathway may be another mechanism explaining the increase in apoptosis and reduction in malignant behavior of AGS cells. Conversely, downregulated Cl− recycling and increased pHi levels may explain the malignant behavior in cancer cells with downregulated Slc26a9 and AE2 expression.Additional insight into the molecular mechanisms that may enhance malignant cell behavior in the absence of Slc26a9 was obtained by studying the expression of the Cld loader . We found loader . This dept cells . A high pt cells , as wellpt cells . A decreApart from a potential specific function of the deleted Slc26a9 gene for gastric epithelial function, the loss of parietal cells and the ensuing hypo/achlorhydria that are uniformly present in many mouse models of oxyntic atrophy/metaplasia and neoplasia, will initiate a cascade of events that are conducive to neoplasia development. We found that Slc26a9 deletion-mediated parietal cell loss altered, not only the expression of markers of gastric epithelial cell differentiation, but also that of multiple genes that encode ligands secreted by parietal cells and function as critical growth factors in the gastric mucosa, including SHH, Notch4, TGF-α, ARGE and HB-EGF . Impaire− channel in parietal cells during acid secretion and may be involved in the regulation of cellular growth and survival. Additional research into the molecular regulation of Scl26a9 appears necessary to unravel its exact function in organs that express this molecule.In summary, we found that Slc26a9 deletion leads to dysregulated differentiation of stem and progenitor cells in an inflammatory environment that results in gastric neoplasia in mice. Multiple signaling pathways that are related to the regulation of cell proliferation, apoptosis and differentiation, as well as barrier integrity , 64, 65 Supplementary file1 (JPG 703 KB)Supplementary file2 (JPG 3271 KB)Supplementary file3 (JPG 1020 KB)Supplementary file4 (JPG 541 KB)Supplementary file5 (PDF 228 KB)Supplementary file6 (PDF 468 KB)Supplementary file7 (PDF 1323 KB)Below is the link to the electronic supplementary material.


Apis mellifera is highly appreciated worldwide because of its products, but also as it is a pollinator of crops and wild plants. The beehive is vulnerable to infections due to arthropods, fungi, protozoa, bacteria and/or viruses that manage to by-pass the individual and social immune mechanisms of bees. Due to the close proximity of bees in the beehive and their foraging habits, infections easily spread within and between beehives. Moreover, international trade of bees has caused the global spread of infections, several of which result in significant losses for apiculture. Only in a few cases can infections be diagnosed with the naked eye, by direct observation of the pathogen in the case of some arthropods, or by pathogen-associated distinctive traits. Development of molecular methods based on the amplification and analysis of one or more genes or genomic segments has brought significant progress to the study of bee pathogens, allowing for: (i) the precise and sensitive identification of the infectious agent; (ii) the analysis of co-infections; (iii) the description of novel species; (iv) associations between geno- and pheno-types and (v) population structure studies. Sequencing of bee pathogen genomes has allowed for the identification of new molecular targets and the development of specific genotypification strategies.The honeybee  Apis mellifera) is of enormous economic and cultural relevance to human society. On the one hand, its service as a pollinator of crops is indispensable to procuring food production worldwide, and on the other hand, it provides subsistence for beekeepers and farmers, and apicultural activity is associated with important social and cultural values. In addition, honey commercialization and export is a source of income for several countries around the world, and constitutes a natural food with many potential health qualities . O. OP. lar by qPCR ,291.M. plutonius DNA detection protocols are particularly useful for the screening of large numbers of samples: (i) a LAMP protocol targeting the gyrB (DNA gyrase subunit B) gene, and (ii) a colorimetric nanoparticle-based detection method. The first method has the advantages of showing a 10 times improved detection limit compared to a seminested PCR based on the 16S rRNA gene, and it does not require special equipment to analyze the results, while the second is characterized by its speed and low cost [In addition to the above-mentioned PCR-based diagnostics, two alternative low cost ,293,294.M. plutonius strains pertaining to CC12 comprise “atypical strains” with less demanding cultivation requirements than those of STs of “typical strains” that pertain to CC3 and CC13. In contrast, strains of CC12 display a higher virulence than those of CC3, while strains of CC13 are avirulent [napA (Na+/H+ antiporter) gene for identification of typical strains (CC3 and CC13) and the Fur (ferric uptake-regulator family) gene, for the detection of atypical strains (CC12), is available  and Iflaviridae , while some are unclassified . Recent advances in metagenomics have allowed us to identify new viruses in bees and other pollinators; including bee macula-like virus (BeeMLV) from the Tymoviridae family, Osmia cornuta nudivirus (OcNV) from the Nudaviridae family, Apis rhabdovirus (ARV-1), A. mellifera iflavirus, and A. mellifera feranovirus, among others [Most bee viruses belong to the families g others ,330.A. mellifera filamentous virus (AmFV) [Most genomes of bee viruses are composed of positive-sense single-strand RNA (+ssRNA). RNA viruses have high mutation rates and often form diverse populations of variants or quasi-species . In conts (AmFV)  and OcNVs (AmFV) .Since bee viruses do not replicate efficiently in cell culture, the effect of viral replication in the host cell cannot be assessed. Thus, infections by bee viruses are classified as overt and covert infections according to the presence or absence of clinical signs, respectively. Covert infection appears when a high replication level of the viral agent is reached in affected tissues or organs, and favors horizontal transmission. In overt infections, on the other hand, vertical transmission is the preferred route of virus spread, and the infection typically persists in the population for many generations. The latter is the most common way viruses infect colonies, but as stated before, under adverse conditions, overt infections may turn into covert infections. In addition, different viral pathogens can infect colonies simultaneously and interact with each other, increasing their pathogenic effect. Diagnosis of bee viruses by conventional methodologies is complicated due to the lack of appropriate cultures, common clinical signs of different viral infections, and the high frequency of multiple infections in the individual bee and/or colony. Thus, detection of bee viruses is mainly carried out by molecular diagnostics, focusing on the amplification of a particular specific sequence region of the viral genome. Nucleic acid amplification by conventional RT-PCR has been widely used for the detection of many bee viruses . MoleculOf note, LAMP has been demonstrated to be a rapid, cost-effective, and robust technology that can be used with crude extracts, avoiding time-consuming purification of nucleic acids. For detection of bee viruses, LAMP assays have only been developed for country-specific SBV outbreaks. The developed LAMP assays target the polymerase or viral protein gene of SBV with a similar analytical sensitivity to RT-qPCRs (reviewed in ).In recent years, RT-qPCR assays have become the preferred technique for bee virus detection due to their high sensitivity and the possibility of multiplexing, quantification and high-throughput processing ,336. MorThe most commonly detected viruses associated with economic losses worldwide are ABPV, BQCV, CBPV, DWV, KBV, IAPV and SBV. Studies describing the presence, abundance or prevalence of these viruses utilize either end-point RT-PCR or RT-qPCR assays, and there is an enormous number of specific primers available designed for this purpose. In the context of this review, we will focus on newly developed molecular approaches and on recently reported field studies applying previously developed assays. In Iflavirus, (+)ssRNA. Three variants of DWV have been described, comprising DWV-A, DWV-B , and DWV-C. DWV-C variants group all the sequences resulting from recombination between DWV-A and DWV-B [The deformed-wing virus is one of the main viruses affecting the honeybee. As mentioned above, DWV belongs to the genus nd DWV-B ,340,341.V. destructor functions as a highly efficient vector within the honeybee colony [A. mellifera . Virus infection is associated with the appearance of bees with deformities in the wings and results in a shortened life expectancy, manifested at the colony level by a progressive decrease in the size of the population [DWV spreads via different transmission routes and e colony ,342. Thipulation ,344.The most common method for DWV detection is RT-PCR. A variety of primers have been described that amplify different regions of the virus genome encoding structural and non-structural proteins. Most of the amplification primers target the RNA-dependent RNA polymerase gene (RdRp) ,342,345.Recently, a real-time, micro-scale, chip-based PCR system allowing for rapid detection of viral RNA was described. This methodology amplifies a RdRp region through the use of DNA chips, a technique that identifies a specific nucleotide sequence through DNA hybridization . On the The advancements in sequencing methodologies have provided the possibility of implementing the analysis of complete DWV genomes in such a way that recombination points between the DWV-A and DWV-B variants can be determined, circulating strains originating in different regions can be characterized, and molecular differences between covertly circulating DWV strains or highly virulent viruses can be identified ,354,355.Another virus closely related to DWV is the Kakugo virus (KV), with which it shares 96% nucleotide identity. Both are often considered to be variants of a single species complex ,356. As Iflavirus; its detection by molecular assays such as RT-PCR or RT-qPCR uses specific regions encoding capsid proteins or non-structural proteins such as helicase and RdRp [A. mellifera colonies [Sacbrood virus (SBV) also belongs to and RdRp ,362,363.colonies ,363,364.Iflavirus first discovered in the predatory social wasp. In a recent study testing for the presence of MV by RT-qPCR, 43% of honeybee colonies (n = 69) resulted positive; MV replication could be confirmed in A. mellifera and Vespidae species [Moku virus (MV) is an  species .Sinaivirus) that affect honeybees and were recently detected through metagenomics studies [V. destructor [Varroa infestation levels are not associated, suggesting that this mite is not a vector of LSV [Lake Sinai viruses (LSV) are (+)ssRNA viruses  is a bipartite, (+)ss RNA virus. Although it was the first bee virus isolated and described from honeybees , its famV. destructor could be determined by Celle et al. [Detection and quantification of CBPV based on RT-qPCR assays have been reported. Blanchard et al.  developee et al. ; howeverDicistroviridae (+ssRNA), these viruses normally persist as covert infections within the colony, and are detectable by RT-PCR assays [The acute bee paralysis virus (ABPV), Kashmir bee virus (KBV), and Israeli acute paralysis virus (IAPV), form the ABPV-KBV-IAPV complex. Infection by each of the three viruses shows a similar pathology at the level of the individual bee and the colony. Like most members of R assays .Since ABPV, KBV and IAPV are genetically very similar, there is a potential risk of misdiagnosis including false-positive and false-negative detection, and strain misassignment . To avoiV. destructor is an effective vector of IAPV [IAPV is one of the most studied viruses because it can infect every member of the colony and its detection strongly correlates with colony collapse disorder (CCD) ,381,382. of IAPV .IAPV covert infection induces a broad spectrum of clinical signs including paralysis and body trembling. However, whether the neuron system is the preferred tropism of the virus has not yet been resolved . In a stDicistroviridae and has two ORFs each encoding for a polyprotein, and each preceded by an internal ribosomal entry site (IRES) [IAPV is a positive single-stranded RNA virus that was first described as a distinct pathogen from KBV and ABPV based on its genome sequence in Israel in 2004 . IAPV bee (IRES) . In a phe (IRES)  reportedSeveral RT-PCRs assays have been developed to detect IAPV ,381,383;Tai Truong et al.  developeDistrictoviridae family and has a similar genome structure to ABPV and KBV. Transmission is driven mainly by nurse honeybees that horizontally transfer BQCV from infected cells to healthy larvae in brood food, but queens may also vertically transmit it to eggs. It has been shown that BQCV is associated with the infection of the microsporidian N. apis [Orf2 and the 5′UTR region are most frequently chosen as targets for nucleic acid amplification [One of the most prevalent honeybee viruses is the black queen cell virus, which affects developing queen larvae and pupae during the capped-cell stage. BQCV belongs to the  N. apis . This vification ,389,390.Most studies on bee viruses analyze the presence of several, including DWV, BQCV, ABPV, CBPV, IAPV and SBV. In early developments, Benjeddou et al.  applied V. destructor, Nosema spp., and a diverse set of honeybee viruses. All viral agents were detected by RT-qPCR using either previously reported primers sets [Moharrami and Modirrousta  describeers sets ,389,392 ers sets .In a prospective study, Runcket et al.  investigSimilar results were reported by Cavigli et al. , who shoIn order to identify synergistic interactions between pathogens and/or parasites that may be involved in colony death, D’Alvise et al.  studied V. destructor. Detection of all viruses was performed by RT-qPCR using primers developed by Locke et al. [V. destructor.Molineri et al.  comparede et al. . Five viSeveral multiplex PCR assays have been designed for honeybee viruses ,402,403.® probes with different fluorophores. The main reason for multiplexing is to save cost and time; however, multiplex qPCR is less sensitive than uniplex qPCR and more complex to optimize. Multiplexing is far more effective through a microarray. Numerous honeybee microarrays have been designed, including honeybee immune gene-pathogen arrays [Real-time qPCR can also be multiplexed, usually for the simultaneous amplification of a target and internal reference standards, by using TaqMann arrays ,379 and n arrays . For resn arrays .®) for quantifying ABPV, BQCV, DWV, VDV1 and SBV VLs in naturally infected honeybees with and without clinical signs. After analyzing the VL distribution in both groups, it was possible to define a threshold that allowed for differentiating between covert and overt infections.Real-time PCR assays allow for the quantification of viral load (VL) and determination of thresholds at which clinical signs are observed, so that VL levels can be compared between different seasons or between healthy and diseased bees. Correspondingly, the VLs of six honeybee viruses  have been determined and compared in adult honeybees with and without clinical signs. Two newly developed RT-qPCR assays for the detection of LSV3 and SBV were applied in this investigation. Importantly, statistically significant differences between the VL of positive samples were identified between healthy and clinically affected honeybees for ABPV, CBPV, DWV, and SBV, while for BQCV and LSV3, no statistical differences were observed . MoreoveV. destructor-parasitized pupae of known patrilines were determined at different levels of mite infestation. The results showed different patterns of intra-colonial variation for both variants of DWV, suggesting more complex spatiotemporal dynamics of bee virus infections than was previously thought [The pattern of infection of DWV-A and B variants was analyzed at the intra-colonial level by quantification of bee virus RNA in pupae and larvae using RT-qPCR. To this end, VLs from  thought .The development and optimization of high-throughput sequencing technologies make it possible to sequence the genetic material of the entire community of viruses present within a host, without any prior knowledge of viral genome sequences. This approach is known as viral metagenomic analysis. Metagenomics allows for a better characterization of viral diversity and accelerates the identification of novel viruses affecting honeybees and other pollinators ,411,412.Tymoviridae family, A. mellifera flavivirus (AFV) and A. mellifera nora virus 1 (ANV) [Rhabdoviridae: A. mellifera rhabdovirus-1 (ARV-1) [A. mellifera rhabdovirus-2 (ARV-2) [Recent studies have reported the identification of additional honeybee-infecting (+)ssRNA viruses including the bee macula-like virus (BeeMLV) of the  1 (ANV) . In addi (ARV-1)  and A. m (ARV-2) .Secoviridae, ; (ii) a novel (+)ssRNA virus within the family Nodaviridae, (iii) a tymo-like virus, (iv) A. mellifera Rhabdovirus 1 (ARV-1) (family Rhabdoviridae), (v) a partiti-like virus (family Partitiviridae), and (vi) a circo-like virus (family Circoviridae)Galbraith et al.  performeA. m. ligustica, A. m. syriaca, A. m. intermissa, and A. cerana and their respective V. destructor mites, were analyzed by RNA metagenomics to compare the composition of their viral populations. In this study, two novel viruses: Varroa orthomyxovirus-1 (VOV-1) in A. mellifera and V. destructor, and a Hubei like-virga virus-14 homolog in V. destructor were reported, as well as some recently described viruses: ARV-1, BRV-1, VDV-2, and VDV-3. by [Viromes from the Western honeybee subspecies DV-3. by . It can In this article, we have presented an overview of available molecular diagnostics and genotyping tools for the detection and analysis of the main pathogens of the Western honeybee. This overview should assist in the selection of adequate tools for planned research and inform on a potential lack of molecular tools that would be important to be developed.T. mercedesae), and (vi) eradication programs or rapid and cost-efficient screening of hive products for pathogen contamination .The available molecular tools are indispensable for: (i) in-depth investigations of still-understudied or emerging bee pathogens, (ii) detection of as-yet-unknown or cryptic species and elucidation of their strain composition, (iii) studies of coinfection with particular bee pathogens that synergistically increase pathogenicity, (iv) worldwide surveillance of the most virulent pathogens, (v) tracing the source of outbreaks, (v) a rapid response to emerging pathogens (e.g., Furthermore, the presented information is aimed at facilitating an integrated view on the particularities of each pathogen and pathogenic group and interacting effects between them, as well as their commonalities. On the other hand, it allows for comparing and contrasting the molecular diagnostic approaches used to study these pathogens and pathogenic groups.Many of the pathogenic agents dealt with in this review have been introduced into the managed population of the Western honeybee through man-made upheavals or by trading of bees. As evolutionary adaptation of honeybees to introduced alien pathogens is an extremely slow and gradual process, human intervention is needed to prevent their further distribution and to minimize their damage on the honeybee population. The availability of reliable diagnostic methods for the sensitive and specific detection of bee pathogens is the first step needed in the efficient implementation of control measures.


Lassa fever is an acute viral haemorrhagic disease caused by the Lassa virus (LASV). It is endemic in West Africa and infects about 300,000 people each year, leading to approximately 5000 deaths annually. The development of the LASV vaccine has been listed as a priority by the World Health Organization since 2018. Considering the accelerated development and availability of vaccines against COVID-19, we set out to assess the prospects of LASV vaccines and the progress made so far.et al. (2019) and searched for new vaccine candidates through Google Scholar, PubMed, and DOAJ from June to July 2021. We searched the articles published in English using keywords that included “vaccine” AND “Lassa fever” OR “Lassa virus” in the title/abstract.We reviewed the progress made on twenty-six vaccine candidates listed by Salami Thirty-four candidate vaccines were identified – 26 already listed in the review by Salami et al. and an additional 8, which were developed over the last seven years. 30 vaccines are still in the pre-clinical stage while 4 of them are currently undergoing clinical trials. The most promising candidates in 2019 were vesicular stomatitis virus-vectored vaccine and live-attenuated MV/LASV vaccine; both had progressed to clinical trials.Despite the focus on COVID-19 vaccines since 2020, LASV vaccine is under development and continues to make impressive progress, hence more emphasis should be put into exploring further clinical studies related to the most promising types of vaccines identified. Mastomys natalensis, are the virus’s natural hosts. The disease is primarily transmitted to humans through contact with infected rats’ urine or feces . Human-to-human infections and laboratory transmission can also occur, particularly in health care settings where infection prevention and control measures are insufficient . LF is endemic in the West African countries of Ghana, Benin, Guinea, Liberia, Mali, Sierra Leone, and Nigeria ; an estimated 300,000 cases occur each year in this region, resulting in 5,000 deaths annually . The number of sporadic cases occurring outside of the endemic regions within and outside of Africa is increasing due to an increase in international travel. In the United States, the United Kingdom  and Sweden , these imported cases have been reported.Lassa fever (LF) is an acute viral hemorrhagic fever caused by the Lassa virus (LASV), a single-stranded RNA virus of the Arenaviridae family. The virus was first isolated in Nigeria in 1969 . Rodents, particularly et al., 2019). Fever, fatigue, haemorrhage, gastrointestinal symptoms , respiratory symptoms , and neurologic symptoms  are all common clinical manifestations of the disease. However, cases of LF, especially in endemic areas, may remain asymptomatic . In 2018, Nigeria had the highest number of cases of Lassa fever with 171 fatalities reported from the 633 confirmed cases . Currently, there are no vaccines or effective drug therapies for the prevention or treatment of LF. Treatment with intravenous ribavirin has however been shown to reduce mortality and morbidity from LF if started within the first week of onset.Lassa fever has an incubation period of 3–21 days. The early-stage disease is similar to other febrile diseases such as malaria and is usually only suspected after haemorrhagic symptoms develop in the late stages , thus, a preventive vaccine is a critical public health need in endemic areas, particularly to protect health care providers, who are frequently the most at risk of exposure , Global efforts are being made to develop new vaccines for the control of diseases of global health importance . Lassa virus (LASV) vaccine development has been identified as a top priority by the World Health Organization (WHO) and the Coalition for Epidemic Preparedness Innovations (CEPI). A WHO Target Product Profile (TPP) of LASV vaccines was developed in June 2017 to aid the development of candidate vaccines for clinical evaluation. The TPP outlined the desired characteristics of potential LASV vaccines in two scenarios: preventive use and reactive/outbreak use. This led to an increase in LASV research, which has facilitated the development of vaccine candidates with pre-clinical proofs of concept that could potentially reduce illness, disease outbreaks, and deaths in humans .Lassa fever, one of the viruses causing severe haemorrhagic fever in Africa, is a major public health issue in endemic areas. When left untreated, LF can have a case fatality rate of up to 70% . In 2019, Salami et al identified about 26 candidate vaccines for LF, some of which met the WHO criteria. Notably, the live-attenuated measles virus/Lassa virus (MV/LASV) platform was found to provide robust protection against LASV in animal models. Vesicular Stomatitis Virus (VSV)-vectored vaccine is another promising live virus vaccine platform, with at least two leading LASV vaccine candidates based on it . The most promising vaccine candidates were reported by  to be live attenuated recombinant Mopeia virus/Lassa virus (MOPV/LASV) reassortment and live recombinant VSV and vaccinia vectored vaccines.According to the World Health Organization, LF vaccines should be cost-effective and affordable in endemic areas and stable for a reasonable time without extensive cold chain facilities. They should also provide immunity for special populations such as HIV-positive patients, pregnant women, and children. Their efficacy and protection must be at least 3 years; the method of administration should be simple, and a small number of doses should be required to confer long-term immunity . In this study, we will follow up on the 26 candidate vaccines identified by Salami and colleagues in 2019 to identify other potential candidate vaccines, discuss their prospects, their rates of development, and highlight factors affecting the development of Lassa fever vaccines.Most of the advanced vaccine candidates are expected to undergo human trials soon, though the clinical testing of these products and their deliveries to the most in-need population were reported to present significant problems  databases between June 23et al. (2019), 26 vaccines currently under development have been reviewed to check the present stage of development, target, and development partners. These are presented, along with progress made since then, in In the review by Salami et al., 2019).Vesicular Stomatitis Virus (VSV) is a virus infecting humans and animals and is an excellent candidate as a pseudovector. Five key proteins are encoded by the genomic RNA: phosphoprotein (P), glycoprotein (G), large protein (L), matrix protein (M), and nucleoprotein (N). Recombinant VSV equipped with a reporter gene instead of a VSV G gene in their genome makes it easier to evaluate infectivity in the study of viral entry, including the identification of viral receptors .This vaccine has demonstrated usefulness in cynomolgus monkeys in an immune response against filoviruses including Ebola virus (EBOV), Sudan virus (SUDV), and Marburg virus (MARV). Further protection from LASV by a single vaccine is challenging because of several contributing factors. The virus is dissimilar to other species of filoviruses; that is why it has been classified as the et al., 2020a).ML29 is a live-attenuated vaccine (L-AttV) that was shown as a promising candidate in animal studies. Trials demonstrated that a single dose of the vaccine can provide sufficient protection against the virus. Nonetheless, as the ML29 attenuation mechanism is still not entirely understood, researchers have questioned if the vaccine has sufficient phenotypic stability to account for potential future mutations .On June 15, 2021, IAVI was awarded €22.8 million from the European & Developing Countries Clinical Trials Partnership (EDCTP) and the Coalition for Epidemic Preparedness Innovations (CEPI) to conduct Phase IIb clinical trial of a novel vaccine candidate to prevent Lassa fever disease.IAVI’s rVSV∆G-LASV-GPC uses a recombinant vesicular stomatitis virus (rVSV) vector just like the rVSVN4CT1-LASV vaccine. The same platform was used to produce Merck’s highly efficacious Ebola Zaire virus vaccine, Ervebo®, recently approved by the European Commission, the United States Food and Drug Administration, regulators in several African countries, and prequalified by the World Health Organization .et al., 2021).YF 17D GPC is based on the success of the Yellow Fever 17D (YF17D) vaccine in protecting humans against flaviviruses. The genetic skeleton for this vaccine candidate has been used for the construction of a bivalent YF17D-based recombinant vaccine, which was engineered by combining LASV and YFV .This vaccine, however, still does not show any immunogenicity in non-human primates. It is therefore necessary to explain and investigate the causes of low immunogenicity prior to considering it an option for the management of LSV in African regions .The reassortant ML29 carrying the L segment from the non-pathogenic Mopeia virus (MOPV) and the S segment from LASV is a vaccine candidate under development. ML29 demonstrated complete protection in validated animal models against a Nigerian strain from clade II, which was responsible for the most severe outbreak recorded in 2018  LASV VLP was immunogenic in mice in the absence of adjuvants, with mature IgG responses developing within a few weeks of the first immunization. These findings highlight the importance of using a VLP platform to develop the best vaccine candidates for Lassa hemorrhagic fever and they call for more research in lethal challenge animal models to establish their protective potential.In a study by Branco et al., 2019).This vaccine is an attempt to develop one vaccine for the 7 strains of arenaviruses namely: Lymphocytic choriomeningitis virus (LCMV), Guanarito virus (GTOV), Junin virus (JUNV), Machupo virus (MACV), Sabia virus (SABV), Lassa and Whitewater Arroyo viruses. This can be achieved by choosing a molecule (epitope) common to them and targeting shared structural parts. HLA-transgenic mice were challenged following the peptide pool immunization and the magnitude of epitope-specific CD8+ T cell response was sufficient to reduce the viral titers. However, it was less extensive when contrasted with the responses found after individual peptide immunization .This vaccine has been designed to protect from both Lassa fever and rabies and was promising in the initial stages of testing. The candidate is based on a weakened, inactivated rabies vector. New studies indicate that LASSARAB injected with a GLA-SE adjuvant causes robust antibody production against both Lassa fever and rabies in guinea pigs and mouse experiments. Animals were protected from disease up to 58 days after administration .Previous research shows that immune responses are not usually proportionally correlated with protection from LSV. New studies, however, showed that high numbers of IgG antibodies which attach to the LSV proteins do in fact correlate with the protection from the virus. Thus, the levels of this antibody have the potential to be used to determine the usefulness and efficacy of the vaccine. The next step is to evaluate the experimental vaccine in nonhuman primates before advancing to human clinical trials . pLASV-GPC encodes the GPC gene of the LF (Josiah strain), and when administered in 3 doses dermally, it protected guinea pigs from LF Virus-associated illness and death. The result was replicated in NHPs. Initially, this has been the most promising vaccine candidate as it is the first to commence human trials .DNA plasmid vaccination involves plasmid expressing gene(s) encoding the antigen(s) being introduced to cells, which stimulate an immune response  research. It offered strong protection in animal models demonstrating that viral replication and stimulation of innate immune responses are fundamental for an effective adaptive immune response in Lassa fever and has formed the basis for previously licensed vaccines .The MV-LASV vaccine candidate is a recombinant live attenuated measles vectored vaccine, presenting antigen of the foreign pathogen which has progressed to clinical trials since Salami, et al., 2018). Exoribonuclease (ExoN) activity in NP of MOPV results in the multiplication of a hyper attenuated strain (MOPVExoN6b) which promotes a strong immune response. The vaccine may result in post-exposure prophylaxis evidenced in previous trials by the 100% survival rate in guinea pigs vaccinated 48h after infection .Researchers have successfully inoculated NHPs against LF Virus using a modified Mopeia virus (MOPEVAC) . It generates non-infectious VLPs containing viral surface proteins which can elicit strong T cell and B cell immune responses, which has allowed VLP-based vaccines to be developed for hepatitis B virus and human papillomavirus . According to Geovax, GEO-LM01 has been validated preclinically and is ready for phase I clinical trials. Vaccination of laboratory mice with LF Virus VLPs (3 doses) via the MVA-VLP-TV vaccine resulted in robust immune responses . According to GeoVax, the MVA-VLP-TV vaccine has been validated preclinically and is ready for phase I clinical trials.GEO-LM01 is based on the Modified Vaccinia Ankara (MVA) Virus-Like Particle (VLP) platform . Existing ChAdOx vaccines such as the ChAdOx1 nCoV-19 Covid-19 Vaccine which shows 70·4% efficacy against Covid-19 highlight the likely success of this blueprint for vaccines .ChAdOx1 Lassa and ChAdOx1-biLAMA circumvent pre-existing immunity to human adenoviruses .The Lassa GPCclamp-based vaccine has completed phase I clinical trials with 99% of vaccinated participants producing a neutralizing immune response. However, it has not progressed to phase II trials due to cross-reactivity in HIV patients.et al. (2019) did not include a few vaccine candidates in their 2019 review. Also, new vaccine candidates have emerged since 2019; their stages of development are reported in the table below.Salami et al., 2018).A small-molecule viral entry inhibitor was developed to protect against the Lassa virus by targeting the viral envelope glycoprotein. It is currently the only viral entry inhibitor vaccine for the Lassa virus. Since its development, the phase I clinical trial has been completed and reported to be well tolerated .The MVALassaNP was found to trigger both humoral and cell-mediated immunity against the Lassa virus. It was proposed to be protective against the Lassa virus due to its cell-mediated function and also halted disease progression in pigs .The vaccine combines the benefits of a live vaccine with that of a single cycle of replication. It demonstrated protective immunity against the disease in lethal guinea pig models .MeV-NP was developed by generating MeV expressing NP protein. It induced efficient protection in a single dose in monkeys . In low single doses, the vaccine offered complete protection to Hartley guinea pigs . Currently, the vaccine is funded by the Coalition for Epidemic Preparedness Innovations (CEPI) and is still in the preclinical phase.Researchers from the University of Rochester and Schipps Research Institute adopted a model used to develop a recombinant form of the mammarenavirus lymphocytic choriomeningitis virus (rLCMV) using a colon deoptimization glycoprotein (CD GPC ). They incorporated the CD-GPC to develop rLASV-GPC/CD . ChAdOx1-vectored Lassa fever vaccine encodes the full-length Josiah strain LASV GPC sequence (ChAdOx1-Lassa-GPC) and protective efficacy was seen after one single dose was given to guinea pigs . The vaccine was developed by the University of Oxford and Janssen Vaccines & Prevention B.V. and is being funded by CEPI .This vaccine comprises of proprietary chimpanzee adenovirus vector platform (ChAdOx1) expressing the Josiah strain LASV GPC: ChAdOX1- Lassa-GP, this vector is derived from chimpanzee adenovirus Y25 . It encodes the Josiah strain glycoprotein and for the LASV it completely lacks Vesicular Stomatitis Virus (VSV) G protein hence it has been called N4ΔG .The vaccine was designed for Lassa virus (LASV), Ebola Virus (EBOV), Sudan Ebola virus (SUDV) and Marburg Virus (MARV) . It is still in the preclinical phase of development .IN0-4500 was developed by Inovio’s Pharmaceutical and encodes the LASV Josiah strain, GPC gene, funded by CEPI. It was demonstrated to offer 100% efficacy after two immunizations at four sites , twenty-six vaccine candidates were identified in preclinical stages. Since then, three of them have progressed to clinical trials, with one securing funding for Phase 2 trials. Most vaccine candidates have significantly progressed since 2019 with only a few being withdrawn. Out of eight new candidates included in There is substantial progress in the development of LF vaccines using a variety of novel technologies. In the review by Salami The most promising candidates in 2019 were Vesicular Stomatitis Virus (VSV)-vectored vaccine and Live-attenuated MV/LASV have already progressed to clinical trials. Other candidates that have seen progress include Mopeia, Lassa Virus reassortants with various antigens as well as DNA platforms. The review of new candidates has shown that additional eight vaccines have been designed, seven of them currently in preclinical stages. Some of these are already being evaluated for clinical phases due to successful trials on animal subjects. We, therefore, recommend that further studies be tailored around the four types which have successfully progressed to clinical trials.To be used in endemic regions, the vaccine must be cost-effective, affordable, and sustainable according to the WHO’s target product profiles . The vaccine development has been described as crucial by the WHO since LF was identified as a potential risk for a pandemic. Following the problems in vaccine production, particularly that posed by LSV lineage diversity, there has been an increased demand to see clinical candidates for vaccines that can safely target multiple strains of the virus. It is critical that all vaccines based on the LASV Josiah strain are tested across all lineages to ensure universality. To conclude, since 2019 and despite the focus on the development of COVID-19 vaccines globally, some of the Lassa virus vaccine candidates have significantly progressed in both preclinical and clinical tests shaping a profoundly mature pipeline in terms of safety and usefulness.The authors have identified no conflict of interest in this study.CD8+ T cell:Cluster of Differentiation 8-positive T Lymphocytes;CEPI:Coalition for Epidemic Preparedness Innovations;ChAdOx1:Chimpanzee Adenovirus vector platform;COVID-19:Coronavirus Disease 2019DNA:Deoxyribonucleic acid;DOAJ:Directory of Open Access Journals;EBOV:Ebola virus;G:GlycoproteinGEO-LM01:GeoVax Labs Lassa fever vaccine;GLA-SE:Glucopyranosyl lipid adjuvant formulated in a squalene-in-water emulsion;GP1:Glycoprotein 1;GP2:Glycoprotein 2;GPC:Glycoprotein complex;GPCwt:Wild type Glycoprotein;GTOV:Guanarito virus;HIV:Human Immunodeficiency Virus;HLA:Human leukocyte antigensIDRI:Infectious Disease Research Institute;IgG:Immunoglobulin G;JUNV:Junin virus;L:Large protein;L-AttV:Live-attenuated vaccine;LASSARAB:Lassa Fever Rabies Virus Vaccine;LASV:Lassa Virus; LASV VLP Lassa Virus Virus-Like Particle;LCMV:Lymphocytic Choriomeningitis Virus;LF:Lassa Fever;LSV:Lily Symptomless Virus;M:Matrix protein;MACV:Machupo virus:MARV:Marburg virus;ML29:Mopeia/Lassa reassortant;MOPEVAC:Modified Mopeia virus expressing antigens of pathogenic arenavirusesMOPV/LASV:Mopeia virus/Lassa virus;MVA:Modified Vaccinia Ankara;MVALassaNP:Modified Vaccinia Ankara expressing the nucleoprotein from Lassa virus;MV/LASV:Measles virus/Lassa virus;MV-LASV:Recombinant measles virus vaccine expressing Lassa virus antigens;N:Nucleoprotein;NCDC:Nigeria Centre for Disease ControlNIAID:National Institute of Allergy and Infectious Diseases;NIH:National Institute of Health;NHPs:Non-Human Primate Models;NP:Nucleoprotein;P:Phosphoprotein; pLASV-GPCLassa virus glycoprotein precursor genePODS:Polyhedra Delivery System; PubMedPublic Medline;RABV:Rabies Virus;rLCMV/CD:Recombinant Lymphocytic Choriomeningitis Virus Based on Codon Deoptimization;RNA:Ribonucleic acid;SABV:Sabia virusSUDV:Sudan virus;TPP:Target Product Profile;UK:United Kingdom;USAMRIID:United States Army Medical Research Institute of Infectious Diseases;VLP:Virus-Like Particle;VSV:Vesicular Stomatitis Virus;WHO:World Health Organization;YF 17D:Yellow Fever 17D


SARS-CoV-2 infection can lead to severe acute respiratory distress syndrome needing intensive care admission and may lead to death. As a virus that transmits by respiratory droplets and aerosols, determining the duration of viable virus shedding from the respiratory tract is critical for patient prognosis, and informs infection-control measures both within healthcare settings and the public domain.We prospectively examined upper and lower airway respiratory secretions for both viral RNA and infectious virions in mechanically ventilated patients admitted to the intensive care unit (ICU) of the University Hospital of Wales. Samples were taken from the oral cavity , oropharynx (subglottic aspirate), or lower respiratory tract  and analyzed by both quantitative PCR (qPCR) and plaque assay.7) than in saliva (2.2 × 106) or BAL/NBAL (8.5 × 106) and reached >108 PFU/mL in some samples. The longest duration of shedding was 98 days, while most patients (14/25) shed live virus for ≥20 days.117 samples were obtained from 25 patients. qPCR showed extremely high rates of positivity across all sample types; however, live virus was far more common in saliva (68%) than in BAL/NBAL (32%). Average titers of live virus were higher in subglottic aspirates (4.5 × 10ICU patients infected with SARS-CoV-2 can shed high titers of virus both in the upper and lower respiratory tract and tend to be prolonged shedders. This information is important for decision making around cohorting patients, de-escalation of personal protective equipment, and undertaking potential aerosol-generating procedures. Patients on intensive therapy infected with SARS-CoV-2 tend to be prolonged shedders, excreting virus for far beyond the time periods specified in current guidelines, and live virus titers can be extremely high in both the upper and lower respiratory tracts. The coronavirus disease 2019 (COVID-19) pandemic has resulted in a global human death toll of 5.36 million (as of 21 December 2021) . AlthougWe examined upper and lower airway respiratory secretions in mechanically ventilated patients with COVID-19 admitted to the ICU of the University Hospital of Wales for titers of infectious severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and compared these to quantitative polymerase chain reaction (qPCR). We show that infectious viral particles are readily recoverable from saliva and that these patients can secrete extremely high levels of live SARS-CoV-2 from multiple sites in the respiratory tract well beyond the 20-day isolation period currently recommended by the Centers for Disease Control and Prevention (CDC) for patients with severe COVID-19 , and thug) to collect supernatant, or washed with Dulbecco’s modified Eagle medium (DMEM) if no supernatant was present. Subglottic endotracheal tubes are used in many ICUs as they reduce ventilator-associated pneumonia. Subglottic aspirates represent an accumulation of oropharyngeal secretions that accumulate above the endotracheal cuff. Bronchoalveolar lavage (BAL) was undertaken using a disposable Ambu aScope 4 and Broncho Sampler Set (Ambu UK) with lavage of up to 80 mL of sterile saline; alternatively, a nondirected BAL (NBAL) was performed by inserting a suction catheter into the lung until resistance was met and 20 mL of sterile saline inserted and slowly withdrawn. All patients received evidence-based treatment as per published health board or ICU directorate guidelines. Samples were transferred to the BSL3 laboratory and processed within 4 hours. Baseline characteristics and treatments are shown in Saliva was collected using Neutral Salivettes , which were placed against the buccal mucosa for 2 minutes and then spun (2000 × Sample collection (20 October 2020–8 August 2020) was undertaken as a service evaluation to see if virus could be measured in respiratory tract samples as an alternative to reverse transcription–qPCR (RT-qPCR). From 1 February 2021–31 March 2021, participants were enrolled in ENLIST (REC reference 20/YH/0309) and consent taken from a relative or legal representative. Sampling was prospective, and weekly unless clinically indicated otherwise. Inclusion criteria were as follows: (1) age 18 years or older, (2) clinical presentation of COVID-19, (3) PCR positive for SARS-CoV-2, and (4) admitted to the ICU requiring invasive mechanical ventilation. The exclusion criterion was age younger than 18 years.2. Plaque assays utilized VeroE6 expressing angiotensin converting enzyme 2 (ACE2) and transmembrane protease serine 2 (TMPRSS2) to enhance virus entry /mL) to extremely high (>10 PFU/mL) . In cont4 copies/mL, suggesting a “cutoff” for detection of infectious virus. However, among samples containing live virus, genome titers were as low as 102 copies/mL. The correlation was even weaker in BAL and subglottic aspirates, where we failed to isolate live virus from samples containing RNA levels as high as 109 genomes/mL, but successfully isolated virus from samples with genome titers of 103 copies/mL.Previous studies have demonstrated a correlation between qPCR cycle threshold (Ct) value and the chances of recovering live virus from oral swabs, with isolation of live virus becoming more infrequent as Ct values increase. In accordance with this, qPCR was clearly more sensitive than virus isolation across all sample types. However, when virus and genome titers were compared , we obseIn previous data live virus was rarely detected beyond 10 days after symptom onset from oro- or nasopharyngeal swab samples, even among hospitalized patients . The sitWhile this study was underway, the Alpha variant of concern began to spread. We therefore sequenced the Spike gene to determine whether the isolation of viable virus differed based on strain. No clear differences were seen in the longevity of virus isolation. Furthermore, no correlation was observed between viral load and patient outcome.When viable viral and gene copy loads from each patient were compared longitudinally, the highest viral titers across all patients were recorded from subglottic aspiration samples. However, overall, saliva provided a better indication of infection; whenever live virus was isolated from any sample at any time point, saliva at that time point always contained live virus. In contrast, by qPCR, sample type was largely irrelevant for determining positivity. There were, however, differences in viral load by qPCR, with subglottic aspirates often containing higher titers than saliva.7 PFU/mL in some patients. This was not variant specific as individual patients infected by either Alpha, or earlier variants, shed these high titers of infective virus. Our study also highlights the inadequacy of qPCR in determining the point during the infection course when an intubated patient ceases to present an infection risk to hospital staff; this was particularly true for airway and lower respiratory tract samples, where PCR positivity was poor at predicting live virus. Furthermore, in contrast to studies using oral swabs in hospitalized patients [Current National Health Service guidance states that isolation precautions can be discontinued in individuals with SARS-CoV-2 infection 10 days after symptom onset , while tpatients , Ct valupatients .This adds to an increasing body of evidence relating to the extended duration of live virus shedding from ventilated patients with COVID-19. van Kampen et al  grouped 6 and 4 × 106 PFU/mL in nasopharyngeal swabs from 2 patients [Only 1 other study has titrated live virus from clinical samples. Differences in sample and cell type can alter viral titer readouts; nevertheless, this study demonstrated titers of 5 × 10patients . Thus, dpatients , 24. In patients . Howeverpatients , 25–27, In a proportion of samples, genome titers were lower than live virus titers. This likely reflects the difficulty of extracting RNA from a highly proteolytic sample, and the need to process the sample to exclude carry-through inhibitors—problems that are reduced in nasopharyngeal swabs that most studies use. All qPCR reactions were controlled by RNase P to ensure that inhibitors did not affect results, and this is reflected in the fact that nearly all samples were positive for viral RNA. Nevertheless, the higher processing requirements, and the lability of RNA, may result in the genome copy number being an underrepresentation of the in vivo situation. Despite this, the genome load in our cohort was similar to those previously reported from oropharyngeal  and saliOur study demonstrates that qPCR is not a robust indicator of viable viral shedding in critically ill patients, irrespective of sample type. Patients in the ICU infected with SARS-CoV-2 tend to be prolonged shedders, excreting virus for far beyond the time periods specified in current guidelines, and virus titers can be extremely high in both the upper and lower respiratory tracts. This may be a consequence of infection-induced immunosuppression and/or the use of steroids and/or interleukin 6 (IL-6) blockade to limit tissue damage. This information is important for decision making around cohorting patients, de-escalation of personal protective equipment, and undertaking potential aerosol-generating procedures, particularly given the threat of new variants such as Omicron that have higher transmission rates and greater vaccine escape potential. It also supports the continued use of oral antiseptics in these patients; antiseptics such as chlorhexidine are used routinely to reduce ventilator-associated pneumonia . These mClinical Infectious Diseases online. Consisting of data provided by the authors to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the authors, so questions or comments should be addressed to the corresponding author.Supplementary materials are available at ciac170_suppl_Supplementary_DataClick here for additional data file.ciac170_suppl_Supplementary_LegendsClick here for additional data file.