Dreissena rostrformis bugensis) as a sentinel species for nanoplastic detection is reported. Under laboratory experimental conditions, mussels ingest and retain fluorescent polystyrene (PS) beads with carboxylic acid (—COOH) termination over a size range of 200–2000 nm. The number of beads the mussels ingested is quantified using fluorescence spectroscopy and the location of the beads in the mussels is imaged using fluorescence microscopy. PS beads of similar size (1000–2000 nm) to mussels' preferred food are trafficked in the ciliated food grooves of the gills. Beads of all sizes are observed in the mussels' digestive tracts, indicating that the mussels do not efficiently reject the beads as unwanted foreign material, regardless of size. Fluorescence microscopy shows all sizes of beads are concentrated in the siphons and are retained there for longer than one month postexposure. Combined atomic force microscopy–infrared spectroscopy and photothermal infrared spectroscopy are used to locate, image, and chemically identify the beads in the mussel siphons. In sum, these experiments demonstrate the potential for using mussels, specifically their siphons, to monitor environmental accumulation of aquatic nanoplastics.Here, a set of experiments to assess the feasibility of using an invasive and widespread freshwater mussel ( Can quagga mussels (Dreissena rostriformis bugensis), a widespread and invasive freshwater species that alters local ecosystems, act as a sentinel species for detecting nanoplastics? In the laboratory, mussels ingest and retain 200–2000 nm fluorescent polystyrene beads, which are in the size range for the mussels' preferred food and are trafficked like food in the ciliated grooves of the gills. Dreissenid mussels filter between 1 and 7 L  of water per day,Dreissena polymorpha), PS microbeads were concentrated in the tissues, gut lumen, and hemolymph after 6 d of exposure.For these reasons, the environmental concentration of nanoplastics has been difficult to quantify, and even the environmental concentration of microplastics is low (≈1–10 particles per 100–1000 L) for the analytical techniques necessary to use for nanoplastics.To that end, we carried out experiments using quagga mussels collected from offshore regions of the Laurentian Great Lakes, USA. Our goal was to assess the extent to which mussels ingest and retain nanoscale PS beads—an appropriate model material, as PS  is commonly found in aquatic environments. We designed a series of proof‐of‐concept studies to determine where to look in the mussel body for retained environmental nanoplastics in mussels collected in situ. We exposed the mussels to nanomolar concentrations of carboxylic acid‐terminated fluorescent PS beads. The concentrations of nanoplastics used in this study are likely higher than those expected in open water, but are likely comparable to areas near the outflow of wastewater treatment facilities.7–108 beads, which in addition to impacts on mussel health, raises concerns of bioaccumulation.These results are particularly interesting when viewed in context of previous work by Morton22.1FigureNannochloropsis fed to the mussels is 1500–2000 nm in diameter and given at a concentration of about 0.1 to 0.001 × 10−12m. With the exception of the images with 2000 nm beads, the images shown in Figure −12m. We also completed 24 h dosing studies with 200 and 1000 nm beads at 0.1 and 0.01 × 10−12m . At these lower concentrations, little to no fluorescence was evident in most of the organs, especially with the 200 nm beads. At 0.01 × 10−12m with the 1000 nm beads, substantial fluorescence was evident in the siphons, a phenomenon explained in more detail below.Particularly with the 1000 nm PS beads, fluorescence microscopy demonstrated that the mussels move the beads through the gills in the same manner as described for food particles Figure1.34 Fi8 ± 3 × 108 1000 nm PS beads dosed at 1 × 10−12m (6 × 1010 PS beads in the culture water). With 200 nm beads at 1 × 10−12m, two mussels took up an average of 5 × 109 beads. When exposed to 2000 nm beads at 0.01 × 10−12m (6 × 108 beads in the water), three mussels each took up an average of 1 × 107 ± 6 × 105 beads. Individual variation in filtering rate could explain the difference in the number of beads taken up. It should be noted that the mussels in each treatment group were housed in the same beaker and were, therefore, in competition for the same beads. It is possible that intense filtering by one mussel could have influenced the uptake by other mussels. However, assuming relatively similar uptake and filtration rates by all three mussels in the beaker, the mussels would have cleared the water if they retained all the beads they took up.Fluorescence spectroscopy was used to quantify the number of PS beads the mussels took up . Overall, the mussels ingested and retained one to two orders of magnitude fewer beads after 24 h as compared to the PS bead concentration in the culture water. In 24 h, three mussels took up (mean ± S.D.) 6 × 10−3, making them only slightly negatively buoyant. Due to the dye, the PS beads are brightly colored, and we can observe when they settle, for example, in the sample bottle. We did not observe settling over the time courses of the experiments (24–72 h). Furthermore, mussels create their own microcurrents when filtering and may have had the ability to stir up any settling beads.Another point we considered is that the beads may settle and sediment over time. However, it is likely to only have a minimal effect, if any at all, on the experiment and results. The density of the PS beads is 1.05 g cm−2 in surface water.−2.−1, it would take 4.5 million years for a mussel in the wild to reach a concentration of 108 beads. Therefore, if nanoplastics concentration is on the same order as reported microplastic concentration, uptake is unlikely to cause problems or be a pressing concern. If local nanoplastic concentrations are higher, either due to release from waste water treatment plants, higher concentrations in the benthos where mussels reside, or as a result of microplastic degradation or fragmentation,5 1000 nm particles or 1.0 × 109 50 nm particles. This analysis still assumes one microplastic in 100 L, but locally higher microplastic concentrations could result in very high concentrations of nanoplastics. If the nanoplastic concentration is on the same order as the microplastic material load, bioaccumulation of nanoplastics could occur depending on relative uptake versus excretion rates (as discussed in more detail below).These data raise the question of whether mussel uptake of nanoplastics is a concern at environmental levels of nanoplastics pollution. A 2016 study by Sutton et al. reported an average of 700 000 microplastic particles kmMussels use particle sorting mechanisms at several stages along the digestive tract, and dreissenid mussels can filter particles larger than 700 nm out of the water.These processes of isolating and excreting unwanted particles are not perfect. It has been reported that once particles are ingested into the gut of the blue mussel, they can translocate into the hemolymph and remain in the circulatory system for over a month and a half.With this previous work as context, the physiological pathways by which the PS beads are moving through the mussels are unclear. The presence of the beads in the rectum could be due to 1) rejection in the stomach; (2) rejection in the digestive gland after phagocytosis and being determined indigestible; or (3) transport all the way through the digestive tract as food. Regardless, our results demonstrate that mussels are not immediately able to reject PS beads in their particle sorting processes. The retention of 200 nm particles in the rectums of the mussels  summarizes the clearance studies and Figure S4 (Supporting Information) shows exemplar images of the feces. Figure−12m were dissected after clearing for 20 d , so the shorter clearing time is not necessarily surprising. However, as shown in Figure −12m. At this time, fluorescence signal from the 2000 nm beads was minimal in the other organs . The mussels dosed with 200 nm beads were dissected after clearing for 21 d . In general, the mussels were able to clear a majority of the beads taken up in 24 h . In the 200 nm studies, five mussels retained an average of 5 × 105 to 107 beads were retained in the mussels. It is possible that given enough time the mussel would be able to clear more of the beads. But, even this level of retention raises concerns about biomagnification of nanoplastics up the food chain.Despite the high clearance rates for all bead sizes , at least 102.3−12m, the mussels did ingest ≈2–10× more 1000 nm beads over 3 d as compared to a 24 h exposure (109 beads vs 108 beads). With 1000 nm beads at 0.1 × 10−12m, the mussels ingested comparable numbers of beads (≈108) whether or not extra beads were added every 24 h. Similar trends were observed in a group exposed to 2000 nm beads at 0.01 × 10−12m. When new 2000 nm PS beads were added every 24 h, the mussels took up 3 × 107 and 1 × 108 beads over 72 h. This is only slightly higher than the 24 h exposure, which resulted in uptake of 1 × 107 PS beads . Calculation of the concentration of beads in the mussels revealed that, in general, little to no bioaccumulation occurred.We carried out this study to assess the extent to which the mussels bioaccumulate the beads—that is, increase the internal concentration of beads as compared to the bead concentration in the water. The results are summarized in Table S3 (Supporting Information). At 1 × 102.4In both the uptake and clearance studies, the PS beads concentrated in the siphons  are low enough such that isolating and characterizing them presents a substantial analytical challenge. As our results presented here demonstrate, retention of beads in the siphons of filter feeders is the best concentration mechanism we have found to date. In particular, the accumulation and retention of high concentrations of plastics in mussel siphons provides a unique handle by which to identify and characterize environmental exposure to aquatic plastics. However, environmental plastics are unlikely to be fluorescent like the beads used in these laboratory studies and high‐throughput, high‐confidence detection and quantification remains a paramount challenge.2.5Figure−12m. The IR spectra of the PS beads in the siphons show characteristic PS signals at 1452 and 1492 cm−1. Beads could not be identified in the mussel siphons exposed to lower concentrations of beads. It is possible this is not due to the absence of beads entirely but rather because the randomly chosen imaging locations did not include beads. AFM can only sample a small portion of a  surface at a time, in the case of these images 20–30 µm. It would be prohibitively time consuming to image the entirety of the mussel siphons, so best attempts were made to achieve representative imaging of the samples. We imaged ≈6–10 locations per mussel siphon depending on how easy it was to locate the beads.The ability to detect PS beads using morphological and chemical means is necessary for in situ specimens because environmental nanoplastics are likely not fluorescent. AFM‐IR allowed for both morphological and chemical characterization of 1000 nm PS beads in the mussel siphons. Beads were easily identifiable in the AFM images Figureb in the One of the characteristics of AFM‐IR is that the IR analysis includes only a small sample volume at each location, usually 10–30 nm laterally and 30–60 nm vertically. This means that in many cases the technique only provides chemical characterization data on the surface layer of a sample. If the PS beads are embedded under the top layer of siphon tissue or covered in biofilm, as is likely the case with environmental micro‐ and nanoplastics, their characteristic chemical signatures may not be observable by AFM‐IR because of the technique's limited vertical sample volume. What would likely be detected by AFM‐IR is the chemical signatures of the top layer of tissue of biofilm and not the chemical signatures of the PS beads. In general, the IR spectrum of tissue is similar to a “standard” protein spectrum with the predicted amide I, II, and III bands. This is exactly what we observed.−1 are very strong. These AFM‐IR and PTIR analyses illustrate the potential of these techniques to identify and characterize environmental nanoplastics in mussels.PTIR spectroscopy addresses the challenge of characterizing the chemical signatures of material below the biofilm and may prove a better technique for studying environmental nanoplastics. This technique achieves sub‐micrometer IR resolution and detects signal over a much larger volume. The interference of biofilms and convolution of the PS signals with the siphon tissue signals was avoided, as shown in Figure As presented in this report, taking advantage of accumulation in sentinel filter feeders appears to be the most promising concentration method of environmental nanoplastics to date. In particular, we demonstrated that PS beads concentrate in the siphons, which provides guidance on finding and detecting nanoplastics in environmental samples. We are currently carrying out studies looking for nanoplastics in environmental mussels collected from known polluted “hot spot” sites and reference sites.The field of research on aquatic nanoplastics is largely undeveloped because of the difficulties in isolating the polluting material. The vast majority of peer reviewed data on aquatic sub‐millimeter plastics results from plankton net trawls capturing particles in the size range of tens to hundreds of micrometers. It is likely that these studies underestimate the concentration of aquatic nanoplastics. The existing work in this area has shown that nanofibers are likely much more prevalent than particulate nanoplastics,3Materials: All materials were purchased from commercial sources and used as received, unless otherwise noted. FluoSpheres (fluorescent PS beads) containing a red dye (580/605 excitation/emission) and carboxylate‐modified surface were purchased from ThermoFisher Scientific. The following combinations of bead concentrations and sizes were used: 1 × 10−12m for 200 nm; 1 × 10−12m for 1000 nm; and 0.01 × 10−12m for 2000 nm. Due to the cost of the 2000 nm PS beads, all of the experiments with this bead size were performed at 0.01 × 10−12m, instead of 1 × 10−12m.Mussel Source and Mussel Husbandry: Quagga mussels were harvested by National Atmospheric and Oceanic Administration vessels using a Ponar grab from sites in Lake Michigan  and Lake Huron . The mussels were packed in wet paper towels, transported in coolers, and then transferred to 38 L glass holding tanks. The culture media for the mussels is a simplified hard water variation of the COMBO media previously described.2 · 2H2O: 55.14 g L−1; MgSO4 · 7H2O: 55.45 g L−1; and NaHCO3: 63.0 g L−1. Mussels were fed RotiGrow Nanno (Nannochloropsis) . An algal food solution (1 mL of RotiGrow diluted in 1 L of mussel media) was administered to the mussels in the holding tank dropwise via a feeding bag. This gradual addition prevented the food concentration from getting too high, which can cause the mussel gills to clog and therefore interfere with filtering. The holding tank mussels were fed three times per week, unless their tank still appeared cloudy and green on a feeding day–at which point that day was skipped to give the mussels time to clear the water. The mussels were kept in the holding tanks until selected for experimental trials, for a maximum of 45 d.Laboratory Uptake Experiments—24 h Exposure, General Procedure; See Noted Figures and Tables S1–S3 (Supporting Information) for Details on Mussel Numbers, Replicates, and Bead Concentrations: The laboratory uptake trials were conducted using groups of three mussels housed in beakers containing 100 mL of culture media. Before adding the mussels to the beakers, the target size and concentration of fluorescent PS beads were added, as well as 2 mL of the food solution (to promote active filtering). Three mussels were selected at random from the holding tank and placed in the beakers containing the PS beads. All experiments were conducted at room temperature (22 °C). After a 24 h exposure to the PS beads, the mussels were treated in one of the following ways, depending on the experiment. Dissection of Mussels and Fluorescence Microscopy: Three beakers containing three mussels each—nine mussels total—were exposed to one of three PS bead sizes . In order to quantify plastic beads within all tissues in aggregate, the mussels were digested following protocols modified from Dehaut et al.m HCl. A sample from the digestate solution was analyzed by fluorescence spectroscopy. The analysis of this subsample was extrapolated to the entire digestate solution.Bead Clearance: Beakers of three mussels each were exposed to one of three PS bead sizes  was used to quantify the mean fluorescence intensity in each of the feces images and boxplots produced to show the trends. Regions of interest of 100 × 100 pixels were used. The results were compiled into box plots, demonstrating the trend of decreasing fluorescence in the feces as the mussels cleared the beads.To assess how long the plastic beads were retained in tissues after exposure, six groups of three mussels each were rinsed five times with culture media. Each group of three mussels was then placed in its own clean beaker containing 100 mL of fresh media and 2 mL of the algal food solution. The mussel feces and pseudofeces were collected using a P1000 micropipette every 24 h for the first 7 d, and then every other day thereafter. The rate of collection was decreased because after the first 7 d the changes in fluorescence over 24 h were minimal. After each collection event, the mussels were given fresh media and given 2 mL of food on their regular schedule of 3× weekWhen the feces were no longer fluorescent, the mussels were dissected and imaged via fluorescence microscopy to look for remaining fluorescent material retained within the mussels. The siphons, gills, and rectums (if they could be successfully dissected separately) were examined for remaining PS beads. After the imaging was completed, the slides were scraped, and the mussels digested as described above for quantification by fluorescence spectroscopy.Laboratory Uptake Experiments—72 h Exposure: (Four treatment groups of three mussels each. Data reported for 11 mussels.) These laboratory uptake experiments were carried out under the same initial conditions as described in the 24 h experiments, but the total exposure time was extended to 72 h. The mussels were dosed at concentrations of 1.0 and 0.1 × 10−12m for the 1000 nm beads and 0.01 × 10−12m for the 2000 nm beads. To ensure that the mussels were not clearing all the beads from the beakers and to keep the bead concentration high, experimental groups were included in which the mussels were dosed with the original number of beads at the 0, 24, and 48 h time points. Thus, these groups were offered 3× the original number of beads as were the 24 h exposure groups.Fluorescence Spectroscopy of Digested Tissue: All fluorescence spectroscopy analyses were performed on a Varian Cary Eclipse Fluorescence Spectrophotometer. The excitation wavelength of the red dye in the PS beads was 580 nm and the emission was recorded from 600 to 650 nm. The spectrum was recorded in triplicate and averaged. If the mussel digestate was cloudy or too concentrated for fluorescence spectroscopy, it was diluted with nanopure water until it was clear and colorless before the measurements were carried out. In these cases, accounted for the additional volume when calculating the concentration of PS beads in the mussels.Calibration curves of fluorescence intensity as a function of concentration were used to calculate the concentration of PS beads in the mussels. To control for potential degradation of the PS beads or the fluorescent dye during the experiment and base digestion process, a control solution of PS beads was concurrently exposed to exactly the same conditions as the mussels . New calibration curves were created for every experiment because of the variations in experimental conditions.Mussels collected immediately following the 24 h exposure were used to quantify average uptake of PS beads. Mussels collected at multiple time points following the exposure period were used to determine the extent to which the mussels were clearing the PS beads. In both cases, fluorescence intensity of mussel digestate was measured and the number of beads detected were quantified against a calibration curve.AFM‐IR of Dissected Mussel Siphons: AFM‐IR is a technique that combines the topographical analysis of AFM with IR spectroscopy. The experiments were carried out on a nanoIR2 . The IR spectrum was generated by analyzing the oscillations of the AFM cantilever, which are dependent on the local thermal expansion of the sample. The resolution of the IR spectra is sample dependent , but generally is ≈30–50 nm.−12, 1 × 10−15, and 1 × 10−18m with 1000 nm beads for 24 h and then allowed to clear in clean beakers for 14 d. The mussel was allowed to clear for 14 d based on the results from the bead clearance studies. At 14 d, the fluorescence level in the feces had dropped below the point of oversaturation, but there were beads remaining in the mussels. The mussels were dissected and the siphons isolated, including the inhalant, exhalant, and the tissue connecting the two. The siphons and connective tissue were visibly pink indicating the presence of beads. The siphons were allowed to dry on glass cover slips at 4 °C and then imaged by AFM‐IR. AFM imaging was carried out on nanoIR2 Contact Mode nIR2 probes . Line scan rates were 1 Hz, and the resolution was 512 pixels/line. Experimental spectra are an average of 32 scans. Savitzky‐Golay smoothing  was applied to the raw spectra scans, which were then normalized with the maximum at 1676 cm−1 and averaged.Groups of three mussels were dosed at 1 × 10PTIR Spectroscopy of Dissected Mussel Siphons: PTIR spectroscopy was carried out on a mIRage IR microscope (Anasys Instruments). This technique achieves sub‐micrometer IR resolution and detects signal over a much larger volume. The same samples analyzed by AFM‐IR were used in the PTIR analyses.Notes on Fluorescent Polystyrene Beads: The PS beads of different sizes have different dye loadings. That is, the bigger beads contain more dye molecules. A given number of beads with a larger diameter will appear brighter than the same number of beads of a smaller diameter. Therefore, the fluorescence intensity in the images cannot be taken as an indicator of the number of beads present; the fluorescence microscopy images provide qualitative information on whether beads are present and where they tend to concentrate in the mussel. This effect is likely observed comparing Figure As discussed, substantial uptake of the 2000 nm beads was still evident at these lower concentrations because (1) there were more dye molecules per bead, and (2) 2000 nm is in the preferred food size range.The authors declare no conflict of interest.Supporting InformationClick here for additional data file.


Crocodylus niloticus) in Zambia. Phylogenetically, the virus belonged to lineage 1a, which is predominant in the Northern Hemisphere. These data provide evidence that WNV is circulating in crocodiles in Africa and increases the risk for animal and human transmission.We detected West Nile virus (WNV) nucleic acid in crocodiles ( Flavivirus, family Flaviviridae. WNV has been reported in a variety of species but is maintained mainly between birds and ornithophilic mosquitoes, with incidental transmission to end hosts, such as humans, horses, and other vertebrates (Crocodylus niloticus) in Israel and Mexico and in alligators (Alligator mississippiensis) in the United States (West Nile virus (WNV), the causative agent of West Nile fever (WNF), is an arbovirus of the genus Up to 9 genetic lineages of WNV have been proposed  to extract total RNA and the OneStep RT-PCR Kit (QIAGEN) to detect part of the WNV genome by using the primer pair WNNY-514 (5′-CGG CGC CTT CAT ACA CW-3′) and WNNY-905 (5′-GCC TTT GAA CAG ACG CCA TA-3′). These primers amplified an ≈400 bp fragment in 2/11 samples tested, which we then used for direct Sanger sequencing. The 2 nucleotide sequences we obtained were 100% identical to each other, and a BLAST analysis (https://blast.ncbi.nlm.nih.gov) showed 99% sequence identity to WNV isolate ArD76986/1990/SN (GenBank accession no. KY703854), which was detected from Culex poicilipes mosquitoes in Senegal . We constructed the phylogenetic tree in MEGA6 (https://www.megasoftware.net) by using the maximum-likelihood method and the Jones-Taylor-Thornton matrix-based model with 1,000 bootstrap replicates. Phylogenetic analysis revealed that Croc110/2019/ZM belonged to lineage 1a (For phylogenetic analysis, we aligned complete polyprotein amino acid sequence of Croc110/2019/ZM and reference sequences from GenBank by using MUSCLE (neage 1a  and was Our study confirmed WNV infection in farmed crocodiles in Africa. Clinical signs and pathological changes in multiple organs correlated with those described for WNV infection in farmed American alligators in the United States (Culex quinquefasciatus mosquitoes (Phylogenetic analysis of the complete polyprotein amino acid sequence of Croc110/2019/ZM grouped the virus in lineage 1a. Previous mosquito surveillance studies in Western Province, Zambia, identified WNV lineage 2 from WNF outbreaks have not been reported in humans in Zambia, but 10.3% of 3,625 persons who participated in a serosurvey were seropositive for WNV in Western and North-Western provinces of the country (Additional information on West Nile virus in crocodiles, Zambia.


During 2016–2017, three rabid terrestrial animals were discovered in the raccoon rabies virus–free zone of Long Island, New York, USA. Whole-genome sequencing and phylogenetic analyses revealed the likely origins of the viruses, enabling the rabies outbreak response (often costly and time-consuming) to be done less expensively and more efficiently. Procyon lotor) since 1960 and endemic to New York, USA, since the 1990s (Rabies has been endemic to the eastern United States in raccoons (Procyon lotor), a river otter (Lontra canadensis), and a domestic cat (Felis catus). Here, we describe our efforts to identify the origins of these rabid animals and our contingency plans to eliminate further cases and restore the rabies virus–free zone status.To identify any breaches in the elimination zone, Nassau and Suffolk County health departments, veterinarians, and wildlife control officers routinely submit animals with clinical signs compatible with rabies for rabies virus testing to the New York State Department of Health Rabies Laboratory . Before 2016, rabies had not been reported in Suffolk County since 2009 or in Nassau County since 2007  (5). DurIn March 2016, a rabid raccoon with neurologic signs was trapped in Hicksville in Nassau County. Real-time reverse transcription PCR  found a river otter on Sound Beach on the North Shore of Suffolk County; the otter was submitted to the Rabies Laboratory of the New York State Department of Health in February 2017. After the otter was determined to be rabies virus positive, our goal was to ascertain by WGS and sequence analysis where the otter was exposed, which would help us determine which supplementary contingency actions were required.In November 2017, a cat with neurologic signs that bit a veterinary staff member in Nassau County was submitted for testing. After determining this cat was positive for rabies virus, we included this case in the study.When the raccoon was originally discovered on Long Island, we had contacted multiple state and federal agencies to discuss contingency plans for containing a possible rabies outbreak. We agreed to limit the contingency actions at that juncture to 12 months’ enhanced surveillance, public health alerts to educate citizens, and reminders about free rabies virus vaccine clinics for domestic animals. Only in the event that other rabid animals were discovered during this time would wildlife trap, vaccinate, and release programs be implemented and oral rabies virus vaccine baits be distributed  charged ≈$250/sample for WGS, we were able to save time and money by sampling animals and ruling out some areas as having local transmission and deescalating the initial plan. Applying WGS to priority samples has previously been shown to be a viable strategy for public health laboratories to use in epidemiologic applications  away in Westchester County. Phylogenetic analysis confirmed the virus variant was consistent with those found in downstate New York (clade 1). Although international pet adoption often garners headlines, domestic animal adoption (Using WGS of an assortment of viruses, we revealed the likely origins of 3 raccoon rabies virus variants from 3 animals that had unexpectedly broken into a rabies-free zone. Through intensified enhanced rabies surveillance and WGS, an expensive contingency plan to distribute baits on Long Island ultimately became unnecessary and was not implemented. This study demonstrates the utility of WGS and phylogenetics as part of a multifaceted rabies investigation with tangible real-world consequences.


Viruses cause epidemics on all major crops of agronomic importance, and a timely and accurate identification is essential for control. High throughput sequencing (HTS) is a technology that allows the identification of all viruses without prior knowledge on the targeted pathogens. In this paper, we used HTS technique for the detection and identification of different viral species occurring in single and mixed infections in plants in Poland. We analysed various host plants representing different families. Within the 20 tested samples, we identified a total of 13 different virus species, including those whose presence has not been reported in Poland before: clover yellow mosaic virus (ClYMV) and melandrium yellow fleck virus (MYFV). Due to this new finding, the obtained sequences were compared with others retrieved from GenBank. In addition, cucurbit aphid-borne yellows virus (CABYV) was also detected, and due to the recent occurrence of this virus in Poland, a phylogenetic analysis of these new isolates was performed. The analysis revealed that CABYV population is highly diverse and the Polish isolates of CABYV belong to two different phylogenetic groups. Our results showed that HTS-based technology is a valuable diagnostic tool for the identification of different virus species originating from variable hosts, and can provide rapid information about the spectrum of plant viruses previously not detected in a region. Viruses cause significant yield and quality losses in a wide variety of cultivated crops. Increasing international travel and the trade of plant material, as well as climate changes, enhance the risk of spreading and introducing new viruses and their vectors into production systems ,2. MoreoMoreover, plant viruses can emerge in crops from reservoir wild plant hosts in which they are often asymptomatic. Spread from the reservoirs into a new environment with the establishment of productive infections and effective between-host transmission mechanism are steps that require emergence to occur . In addiMost of the viruses infecting plants are RNA viruses, whose mutation rate is very high . ApproprThe use of the appropriate diagnostic method is crucial in maintaining healthy material, preventing the spread of the diseases, and carrying out phytosanitary measures. A traditional diagnostic method using transmission electron microscopy (TEM) allows only for the morphological observation of particles in leaf sap derived from infected plants . The staHigh-throughput sequencing (HTS) is a rapidly developing technique, providing novel opportunities for diagnosis and epidemiology. This technique allows for the sequencing of millions of nucleotides in a short time, which enables the detection of the most viral pathogens in the sample . MoreoveIn this study, an HTS-based approach was applied for the detection and identification of different viruses from infected plant material collected in Poland. To this end, various plants from different families were used, including cultivated and ornamental plants, weeds and trees. By following this procedure, we successfully detected new pathogens for Poland, identified mixed infections, and obtained the exact genetic characteristic of the pathogens. With the advantage of HTS methods that have been developed to look for virus-like sequences without the bias of only looking for known viruses, we were able to identify virus species that have not been detected using conventional RT-PCR. Furthermore, the phylogenetic analysis was performed in order to establish the genetic relationships of: melandrium yellow fleck virus, clover yellow mosaic virus and cucurbit aphid-borne yellows virus, identified in this study with others described to date.During the surveys performed in Poland in 2018–2019, 50 samples from plants belonging to different botanical families were collected. In all cases, various disease symptoms, in the form of leaf blade deformations of varying severity, discoloration, chlorosis, necrosis of leaf blade, and growth reduction, were observed . CucurbiNicotiana tabacum L. cv. Xanthi, C. pepo convar. giromontiina and Chenopodium quinoa Willd. In the case of tobacco plants , the symptoms were in the form of leaf blade deformations and chlorosis. V. faba cv. minor after performing the bioassay gave disease symptoms only on C. quinoa, in the form of chlorotic spots (sample 13). Samples derived from R. pseudoacacia were characterised by the presence of local necrotic spots and necrotic ringspots on C. quinoa, and leaf deformation and necrotic ringspots on N. tabacum cv. Xanthi  after inoculation to test plants, the symptoms in a form of chlorotic mosaic on N. benthamiana  positive result of bioassay was observed for 45 original plants. The symptoms observed on particular host species and test plants were summarised in the thamiana d, and lesample 9—.Potexvirus genus, were present  were characterised by the presence of isometric particles of about 30 nm in diameter, whereas in samples derived from ornamental plants (G. jamesonii and C. multiflorum), the presence of isometric particles with a diameter of about 80–100 nm was observed (After the examination of the collected samples under the transmission electron microscope (TEM), the presence of various viral particles, both isometric and filamentous, of different lengths and diameters was observed. In tomato samples, mainly filamentous virus particles about 530 in length, characteristic for the  present a. Additi present d, or iso present b, or filobserved c.R. pseudoacacia, one from S. nigra and one from V. faba cv. minor, no symptoms were observed on the test plants, no particles were observed at TEM, and the RT-PCR assay was also negative. These samples were not subjected to further analysis. In tomato samples, mostly pepino mosaic virus  was detected, but the presence of cucumber mosaic virus  or potato virus Y  has also been noticed , which confirmed the presence of this virus in the tested samples (G. jamesonii and C. multiflorum), the presence of TSWV was observed, with mixed infection with CVB in a sample derived from chrysanthemum , we did not identify any of the tested viruses. In 16 original samples, six of C. pepo convar. giromontiina, two of C. sativus, two of S. lycopersicum, one of R. pseudoacacia, one of V. officinalis, one of S. latifolia, one of R. × prostrata, one of D. belladonna and one of V. faba cv. minor, despite the symptoms on test plants and the presence of virus particles in TEM, we did not identify any viruses using selected RT-PCR tests. These samples, together with three zucchini samples  on V. officinalis plants, and melandrium yellow fleck virus (MYFV)  on S. latifolia plants. The HTS analysis of the third weed (R. × prostrata–sample 16) revealed the presence of turnip mosaic virus  in the sample  and cucumber leaf spot virus . Reads mapped to the CLSV reference sequence (NC_007216) were detected in one sample  and accoThe data analysis of high-throughput Illumina sequences from tomato plants revealed the presence of the PepMV, in complex with tomato yellow ring virus , or CMV .V. faba cv. minor (sample 7), the presence of bean yellow mosaic virus  was detected. In sample 1, which came from R. pseudoacacia, the peanut stunt virus  was mapped to reference sequences. In The obtained results of some of the analysed samples were confirmed by standard Sanger sequencing, which also allows to complement HTS contigs to obtain complete viral genomes. Complete genome sequences of identified viruses using HTS were placed in the GenBank, under the accession numbers: MT153866-MT153870, MT130394 and MT176428.The isolate ClYMV-2018/1 (MT176428) was collected in 2018 in the Wielkopolska region of Poland. The full length genome sequence of ClYMV-2018/1 was compared with the only one available complete RNA sequence of ClYMV isolate originated from Canada (NC001753). The Polish and Canadian isolates shared overall 80.8% nucleotide identity (nt). A comparison of particular open reading frames (ORFs) was also performed. The analysis revealed the following nucleotide (nt) and amino acid (aa) identities: 79.4% and 88.6% for RNA dependent RNA polymerase (RdRp), 82.5% and 90.3% for triple gene block 1 (TGB1), 87.8% and 89.4% for TGB2, 88.7% and 88.1% for TGB3, 83.7% and 92.9% for coat protein gene (CP). In order to obtain the knowledge of the genetic diversity of the ClYMV population, a phylogenetic analysis was performed based on the short fragment (375 nt) of the CP gene of the ClYMV isolate obtained in this study and others described to date. The analysis revealed that the Polish isolate grouped together with the isolate from the United Kingdom . The full length RNA1-3 sequences of the newly detected Polish isolate MYFV–2018/1 were compared with the only one available genome sequence (RNAs1-3) of MYFV (AB444583–AB444585). The nucleotide sequence identity between the Polish and Hugarian isolate collected from Melandrium album was 94.8% for RNA 1, 91.6% for RNA 2 and 91.1% for RNA 3, respectively. For better insight of genetic diversity between both isolates, the particular ORFs were also compared. The analysis revealed the following nucleotide (nt) and amino acid (aa) identities: 94.8% and 98.5% for 1a gene, 91.4% and 95.6% for 2a gene, 91.2% nt and 91.8% for 3a gene, 92.4% and 99.4% for CP.The phylogenetic analysis was performed to obtain an information about the phylogenetic relationships of CABYV isolates from this study with other sequenced isolates of this virus. In the analysis, four Polish CABYV isolates were used; full length genome sequences of three of them were obtained in this study , whereas one (MK059479) was obtained in the previous experiments . The isoIn the present study, we used different diagnostic methods to detect plant viruses from crops, ornamental plants, weeds, trees and shrubs. An important issue in the disease management is the limited availability of appropriate diagnostic methods. In fact, effective detection protocols are required for maintaining healthy planting material. Standard diagnostic tests, despite potentially high sensitivity and specificity, require specific prior knowledge of the target pathogens, and thus are not suitable for detection of unknown or unexpected pathogens. Moreover, their high specificity might limit the detection of different isolates, variants or strains of particular pathogens, which can lead to false negative results ,33. GivePotexvirus genus within Alphaflexiviridae family. Its genome consists of single-stranded plus RNA about 7000 nt in length. The phylogenetic analysis revealed that the Polish isolate is closely related to the isolate from the United Kingdom. The second one—MYFV—belongs to the Bromovirus genus of the Bromoviridae family. Bromoviruses have three positive-sense RNAs as their genome, designated RNA1, RNA2, and RNA3 [M. album ranged between 91.1–94.8% for the genomic RNA1-3. ClYMV and MYFV were detected in weeds that can serve as alternative hosts and potential sources of virus infection. In addition, many wild plants are often infected with more than one viral species at the same time [In this study, we identified two new viruses which originated from weeds: ClYMV and MYFV. The first one, ClYMV infects a broad bean, pea, alfalfa, fat hen, chickweed, and tulips ,35,36. Mand RNA3 . MYFV ocame time . SurprisCucurbitaceae family sampled in Poland are infected with a large spectrum of viruses in mixed infections, including CMV, ZYMV, WMV, CLSV and CABYV. Cucurbits are the major vegetables cultivated worldwide and are affected by more than 70-well-characterised viruses belonging to the main plant virus groups [Aphis gossypii, M. persicae and Macrosiphum euphorbiae). The abundance of CABYV reservoirs found around cultivated fields and the large populations of A. gossypii and M. persicae vectors indicated the potential for virus spread, and represents a serious threat to cucurbit production in Poland. It has been shown that CABYV is one of the most common cucurbit viruses in open-field crops, distributed worldwide [We have shown that plants from the s groups . Most ars groups . In 4 ofs groups . This was groups . The virorldwide . IncreasTospovirus genus: tomato spotted wilt virus and TYRV were observed in tomato crops in Poland [We have also identified the presence of CLSV in complex with other viruses on zucchini plants. This is the second study reporting the presence of this pathogen in our country . The virn Poland . The virn Poland ,7,8,9. Tn Poland . Such ocIn summary, we detected a wide spectrum of pathogens using the HTS-based approach. We have shown that HTS technology is appropriate for the detection of viruses in plants in the absence of prior information on the type of pathogen and its genome structure, or in the case of unclear symptoms of infection. Moreover, we confirmed the potential of HTS for use in the identification of all viruses in mixed infections, including those that could escape detection by RT-PCR. The use of HTS for virus detection will improve the current diagnostic methods, by thoroughly investigating the genomic sequence and the variability of known pathogens, and by designing new diagnostic tools for new viruses.S. lycopersicum, C. pepo convar. giromontiina, C. sativus, V. faba cv. minor), trees and shrubs , ornamental plants , and weeds . On the collected plants, various disease symptoms, in the form of leaf blade deformations of varying severity, discoloration, chlorosis, necrosis of leaf blade, and growth reduction, were observed.During the growing season (June–September) in 2018 and 2019, a total of 50 plants with different symptoms were collected from different regions in Poland. Part of the samples were provided from the Plant Disease Clinic IPP-NRI. The material consisted of various crops  were ground in 2 mL of 0.05 M phosphate buffer (pH 7.2) and the obtained sap was applied on tested plants [All the collected samples were transferred by mechanical inoculation on carborundum dusted test plants: d plants . Plants Subsequently, the presence of the viruses was checked by the standard transmission electron microscope (TEM) procedure. Leaf samples from all the infected plants were crushed in distilled water and applied to Formvar coated copper grids . Then, the grids were dyed with ammonium molybdate (MA) or phosphotungstic acid (PTA) and dried . The preCucurbitaceae family were tested for the presence of CMV, CABYV, cucumber green mottle mosaic virus , papaya ringspot virus , TBRV, WMV and ZYMV. Tomato plants were tested for PepMV, PVY, and CMV, whereas V. faba cv. minor was checked for broad bean true mosaic virus  and BYMV. Ornamental plants and trees were tested for the presence of viruses from the Nepovirus genus using the primers for nepo A, nepo B and nepo C groups. Moreover, gerbera plants were tested on TSWV and chrysanthemum plants on Chrysanthemum virus B  and TSWV. All the primers used in the experiment are summarised in Total RNAs were isolated from all collected plants and from test plants 10 days post inoculation (dpi), using RNeasy Plant Mini Kit , according to the manufacturer’s protocol, and dissolved in 50 μL of sterile water. Then, RNAs were measured fluorometrically using a Qubit 3 fluorometer . The presence of the selected (most common) viruses in each sample was tested using a Transcriptor One-Step RT-PCR Kit , according to the manufacturer’s procedure. Plants from the The next generation sequencing procedure was performed for 20 samples . For HTSThe presence of selected viruses detected by HTS was checked by standard RT-PCR procedure and Sanger sequencing. The presence of the viruses in each sample was checked using a Transcriptor One-Step RT-PCR Kit , according to the manufacturer’s procedure, with a set of specific primers designed based on the consensus sequences obtained by HTS . PCR proThe full-length genome sequence of ClYMV-2018/1 was compared with the one genome sequence of ClYMV available in GenBank NC001753). The comparison of particular ORFs was also performed using the Sequence Identity Matrix in BioEdit software [3. The coThe nucleotide sequences were aligned by codon using MUSCLE algorithm , as implThe genomic RNAs of MYFV-2018/1 isolate obtained in this study were compared with one full-length genome sequence of MYFV deposited in GenBank. The Hungarian isolate of MYFV originated from M. album (AB444583-AB444585). Nucleotide sequence identity of RNA1-RNA3 was determined using the Sequence Identity Matrix in BioEdit software .The phylogenetic analyses were conducted using the gene encoding coat protein (CP) sequences of 37 CABYV isolates, both obtained by us in this experiment, as well as those deposited in GenBank. Moreover, MABYV (MC010809.1) was used as the outgroup. The information about particular isolates  were placed on the tree. The CP nucleotide sequences were aligned by codon using MUSCLE algorithm , as impl


KIFBP). However, the phenotypic range of this syndrome is wide, indicating that other factors may play a role. To date, 37 patients with GOSHS have been reported. Here, we document nine new patients with variants in KIFBP: seven with nonsense variants and two with missense variants. To our knowledge, this is the first time that missense variants have been reported in GOSHS. We functionally investigated the effect of the variants identified, in an attempt to find a genotype–phenotype correlation. We also determined whether common Hirschsprung disease (HSCR)‐associated single nucleotide polymorphisms (SNPs), could explain the presence of HSCR in GOSHS. Our results showed that the missense variants led to reduced expression of KIFBP, while the truncating variants resulted in lack of protein. However, no correlation was found between the severity of GOSHS and the location of the variants. We were also unable to find a correlation between common HSCR‐associated SNPs, and HSCR development in GOSHS. In conclusion, we show that reduced, as well as lack of KIFBP expression can lead to GOSHS, and our results suggest that a threshold expression of KIFBP may modulate phenotypic variability of the disease.Goldberg–Shprintzen syndrome (GOSHS) is caused by loss of function variants in the kinesin binding protein gene ( GOSHS was first described by Goldberg and Shprintzen in 1981, and to date, 37 cases have been reported through clinical diagnosis, with variable severities and additional features, such as iris coloboma, cleft palate/bifid uvula, corneal ulcers, and congenital heart defects , the Semaphorin 3A gene (SEMA3A), and the Neuregulin 1 gene (NRG1) are known to be associated with HSCR risk. Previous studies have already investigated the effect of common variants located in intron 1 of RET in a series of patients diagnosed with a Mendelian syndrome where HSCR is part of the phenotype  is reported in ~70% of patients with GOSHS, but is considered to be a variable feature. HSCR is characterized by the absence of enteric ganglia in the distal colon and occurs in multiple defined syndromes .Sanger sequencing of RET , supplemented with 10% fetal calf serum (Gibco) and 1% penicillin/streptomycin (Gibco). Cells were incubated at 37°C and 5% CO2. For transient transfection, 3 × 105 cells were seeded per well in 6‐well plates. After 24 h, transfection with expression vectors containing either HA‐tagged wild type or mutated KIFBP complementary DNA (cDNA) was performed using GeneJuice® transfection reagent (Millipore), according to the manufacturer's instructions.Human embryonic kidney cells (HEK293) were cultured in Dulbecco's modified Eagle's medium high medium containing 4.5 g/L glucose, 2.5RNA was isolated from HEK293 cells transfected with KIFBP wild‐type (WT) and mutant constructs, using the RNAeasy Kit (Qiagen), according to the manufacturer's instructions. cDNA preparation and quantitative polymerase chain reaction (q‐PCR) were performed, in triplicate, as previously described . Cells were stained for HA using the HA‐Tag antibody  at 1:1500 dilution, and the Cy3 AffiniPure donkey antirabbit IgG, 1:200 dilution . Three wells were fixed for each construct. Cells were imaged on a Leica SP5 confocal microscope.2.7Twenty‐four to 48 h after transfection, cells were washed with PBS and lysed as described before . All data were analyzed using a two‐tailed Student 33.1KIFBP .Patient IE1 is a female with a classical GOSHS phenotype and HSCR. She was found to have a homozygous two base‐pair deletion at c.1694_1695 (NM_015634.3:c.1694_1695delAG) of KIFBP (NM_015634.3:c.1516dupA) was identified in these patients, leading to the appearance of a premature stop in Exon 7 (Accession number: SCV000994838).Siblings PL1 and PL2, are both males with classical GOSHS facial dysmorphism. PL1 has HSCR, where PL2 does not. A homozygous insertion of an A at position c.1516_1517 of KIFBP (NM_015634.3:c.875_990del). Patient UK3 is a male, with classical GOSHS phenotype and HSCR. Patient UK4 is a female and does not have HSCR (accession number: SCV000994839).Siblings UK3 and UK4 were found to have a homozygous deletion of Exon 6 of KIFBP (NM_015634.3:c.790_990del). Both have a classical GOSHS phenotype, but NO1, female, has HSCR where it is absent in her brother, NO2. This deletion is homozygous and has been previously reported , all truncating variants identified in our GOSHS patients are classified as pathogenic Table S2). These variants are also predicted to result in a total loss of KIFBP expression , and one in Exon 7, NM_015634.3:c.1279A>G  is a 10‐year‐old male of Moroccan ancestry, born to consanguineous parents. He had a history of microcephaly and presented with short stature. Brain imaging showed pachygyria and he was affected by demyelinating peripheral neuropathy and perceptive deafness. However, he lacked the typical facial features of GOSHS, had no skeletal symptoms, and had no reported gastrointestinal or enteric nervous system abnormalities. As GOSHS was initially not considered a possible diagnosis due to the lack of characteristic features, whole‐exome sequencing was conducted on DNA from blood collected for diagnostic purposes. This resulted in the identification of two heterozygous missense variants in  is a 10‐KIFBP showed a homozygous missense variant in Exon 3, NM_015634.3:c.565C>T , is a 28‐year‐old female of Cypriot ancestry, born to reportedly nonconsanguineous parents. She had a history of microcephaly, mild intellectual disability, and developmental delay. She presented with short stature, typical dysmorphic facial features with bilateral blepharoptosis, and corneal ulcers. She was diagnosed with HSCR at the age of 3 years. In addition, she had scoliosis, lordosis, pes cavus, as well as mild sensory motor neuropathy with both axonal and demyelinating features. Brain magnetic resonance imaging did not reveal a central nervous system (CNS) abnormality. No copy number variations were detected with array‐CGH. Review of the family history revealed that the patient's younger brother was diagnosed with HSCR and died in the neonatal period from sepsis, following surgery for meconium ileus. There was also a report of a maternal relative who apparently died in infancy and had features suggestive of GOSHS. Sequencing of 3.3To evaluate the effect of the missense variants, expression levels of KIFBP were determined after the transfection of HEK293T cells with constructs expressing the WT and mutant KIFBP cDNA. q‐PCR results showed significantly decreased RNA levels for all mutants when compared with the wild‐type Figure . All mut3.4Tagged WT and mutant KIFBP constructs were overexpressed in HEK293 cells, to determine any effect of the variants in the organization or localization of KIFBP within the cell. The WT protein is seen to have high cytoplasmic expression, as previously described , NRG1, and SEMA3A (Kapoor et al., p = .526, Table In this manuscript, we report nine new patients with variants in KIFBP are known to cause GOSHS. However, there seems to be no correlation between the location of the variant, and the severity of syndromic characteristics (Figure KIFBP (Figure KIFBP that leads to the amino acid substitution of proline by a serine. Patient NL1 is compound heterozygous, and has two different missense mutations in each of his alleles. One leads to the substitution of glutamate by glycine, while the other changes a serine into glycine. Based on the chemical properties of the different amino acids, the most dramatic change is expected to be the one found in patient CYP3. This is because proline is a nonpolar amino acid that is normally present buried within the protein core due to its hydrophobic nature, while serine is a polar, hydrophilic aminoacid, able to form hydrogen bonds. For patient NL1, although the amino acid changes identified lead to the substitution of relatively big amino acids, glutamate and serine, by glycine, the smallest amino acid due to its minimal side chain, they can basically fit into hydrophilic or hydrophobic environments with relatively minor consequences. Prediction tools and conservation scores are shown in Table KIBBP were only identified in this patient after exome sequence, and we cannot exclude that they are just polymorphisms due to the fact that they have both been found in the general population. However, our functional results suggest otherwise, as a reduction of KIFBP expression was detected for the three variants tested (Figure KIFBP can be as damaging as truncating variants, depending on its effect on protein expression levels.LOF variants in s Figure , as theys Figure . In seveP Figure . Unfortud Figure , suggestKIFBP variants, we believe that the accurate classification of GOSHS based on phenotype by a clinical geneticist may be more useful for the family to appropriately meet the needs of the patient, as well as for advising clinical treatment.It has been previously noted that the diagnosis of GOSHS should rely on molecular and genetic findings in place of phenotypic recognition only, due to its similarity with other syndromes (Salehpour et al., All the authors declare that there are no conflict of interests.Supporting informationClick here for additional data file.


In addition, the model deciphered the coinfection of several respiratory pathogens and severe COVID-19 disease afflicted by early variants of the SAS-CoV-2 virus. The high prevalence of these coinfections was explained by the pro-inflammatory and “low Treg” nature shared by respiratory pathogens and severe (“chronic”) COVID-19 triggered by these early variants.In a recent publication, the binary model of chronic diseases was applied to SARS-CoV-2 infection . The effonly if a surge in the blood levels of cytokines like IFN-γ, IL-1, IL-6, TNF-α or IL-10, has been observed; (c) the frequency of coinfection with Respiratory Syncytial Virus, influenza A virus, Influenza B virus, Parainfluenzae, Mycoplasma pneumoniae, Pseudomonas aeruginosa, Haemophilus influenza or Klebsiella pneumoniae is expected to be lower compared to earlier variants [since all these pathogens induce “low Treg” reaction  in South Africa suggested lower odds of severe disease compared to the Delta variant . Similar29 in Soureaction –18. For The author confirms being the sole contributor of this work and has approved it for publication.The views and opinions expressed, and/or conclusions drawn, in this article are those of the author and do not necessarily reflect those of Taro Pharmaceutical Industries Ltd., its affiliates, directors or employees.Author ZE is employed by Taro Pharmaceutical Industries Ltd.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


In low-resource settings, resilience to infectious disease outbreaks can be hindered by limited access to diagnostic tests. Here we report the results of double-blinded studies of the performance of paper-based diagnostic tests for the Zika and chikungunya viruses in a field setting in Latin America. The tests involved a cell-free expression system relying on isothermal amplification and toehold-switch reactions, a purpose-built portable reader and onboard software for computer vision-enabled image analysis. In patients suspected of infection, the accuracies and sensitivities of the tests for the Zika and chikungunya viruses were, respectively, 98.5%  and 98.5%  and approximately 2 aM and 5 fM . The analytical specificities and sensitivities of the tests for cultured samples of the viruses were equivalent to those of the real-time quantitative PCR. Cell-free synthetic biology tools and companion hardware can provide de-centralized, high-capacity and low-cost diagnostics for use in low-resource settings. Double-blinded studies show that paper-based diagnostic tests and a companion portable device designed for use in low-resource settings perform well for the diagnosis of the Zika and chikungunya viruses in serum samples. This public health crisis highlighted the need for rapid and low-cost testing that can be deployed beyond the reach of centralized clinical diagnostic labs. Such centralized labs use real-time quantitative PCR (RT–qPCR) for the detection of pathogens, which, although tremendously sensitive and specific, requires specialized laboratory equipment that is cumbersome and costly. The result is a sparse network of diagnostic hubs that can be difficult to scale during an outbreak, often leading to bottlenecks in testing8. This was the case in the hardest-hit country, Brazil, where RT–qPCR-based diagnostics for the Zika virus were provided by five centralized national reference laboratories, which led to limited access and delays in results10. The circumstance was worsened by overlapping clinical symptoms of the Zika virus with other endemic arboviruses12, cross-reactivity in antibody tests and a lack of portable antigen tests13.The 2015–2016 outbreak of the Zika virus in Latin America transformed the virus into a global concern that infected an estimated 210,000 patients and caused congenital anomalies in thousands of newborns in Brazil alone14. Taken together, the shortfall in diagnostic capacity led to calls for molecular diagnostics that can be used at the point of care (POC) and motivated the development of several new Zika diagnostic technologies, many of which came from the field of synthetic biology17.Although most patients do recover from the Zika virus, the nature of mosquito-borne transmission makes this a disease of poverty, with most infections occurring in peri-urban settings, where standing water is common and public infrastructure is often inadequate. Studies following the 2016 outbreak found that inadequate surveillance and diagnostic systems contributed to spread of the Zika virus, and that social and economic disparity in access to health services exacerbated the challenges faced by vulnerable populations15. Using computationally designed toehold switch-based sensors ) to create an optical signal through enzymatic cleavage of chlorophenol red-β-D-galactopyranoside ) was placed upstream in the workflow to provide detection down to the low-femtomolar range (3 fM)20. Notably, all of the molecular components for the test are independent of the PCR supply chain and, if needed, can be fabricated using the infrastructure of a simple microbiology lab24. These features make this in vitro and biosafe technology well suited for use where there is a need for de-centralized capacity, such as in low-resource settings or during public health crises.We were one of the groups that contributed to the effort for new and portable Zika virus diagnostics15. Here we report field trials for a synthetic biology-based diagnostic using patient samples, and we benchmarked the test performance directly to a US Centers for Disease Control (CDC) RT–qPCR test for the virus25. After optimization of the molecular components, and as part of establishing a PCR-free molecular diagnostic at field sites away from the benches of home laboratories in Canada and the US, we developed a low-cost, computer vision-enabled, automated plate reader  to perform low-volume, high-capacity optical measurements in a 384-well format. Using RNA extracted from the serum of patient samples, we find that the combined PLUM reader and paper-based Zika sensor provide analytical sensitivity and specificity for the Zika virus equivalent to RT–qPCR, with a diagnostic accuracy of 98.5%. We further show that the combination of molecular, hardware and software tools can be used as an adaptive system for the point of need. In a second proof-of-concept effort, we demonstrate that by simply changing the molecular components that confer specificity to the assay , we can detect the chikungunya virus (98.5% accuracy), which is another arbovirus with worldwide distribution that causes significant morbidity28.Until recently, portable diagnostic technologies based on synthetic biology have remained largely untested under field conditions with patient samples. To address this need for validation, we assembled a team of laboratories from five countries to perform a patient trial of our paper-based Zika diagnostic devices onsite in Latin America Fig. 15. Here 15. This started with evaluation of analytical sensitivity using in vitro-transcribed RNA inputs. As previous work has shown, the incorporation of an isothermal amplification step can be used to provide detection of target nucleic acids well within clinically relevant concentrations29. Using this two-step process, we demonstrated detection of target RNA from concentrations as low as 1.24 molecules per µl   provided the best sensitivity.We began by optimizing the original paper-based Zika virus diagnostic workflow for distribution to field sites , the lentivirus contains only a small portion of the Zika viral genome, reducing risk of disease, and has been engineered with safety features that mitigate many of the risks of working with Zika virus, including replication incompetence3 plaque-forming units (PFU) per ml. With the diagnostic assay ready for field testing, the primer sequences, toehold switches and protocols were transferred to laboratories in Brazil, Ecuador and Colombia to establish testing capacity onsite in Latin America.We began by extracting RNA from the lentiviral particles using conventional column purification Fig.  and thenn < 16) and required a laptop and separate incubator for operation22. Moreover, these designs required a dedicated light-emitting diode (LED)/sensor pair (US$6.50) for each sample, which alone would make the deployment of the high-capacity system needed for a patient trial impractical and costly for a 384-well format.The transition of a paper-based diagnostic assay to field-deployable applications requires a field-ready companion device, and so, with no commercial options for low-cost and portable optical quantification, we developed a portable device that provides quantitative and high-throughput measurements of the paper-based Zika diagnostics onsite. Previous efforts developed companion electronic readers; however, the devices had limited sample capacity  were arrayed using a custom-printed circuit board to create a simple and low-cost illumination source for the PLUM reader . To measure absorbance, conventional plate readers directly monitor the intensity of the wavelength of interest and create an absorbance value by inverting the quantified decrease in transmitted light signal , and the RGB channel values for all well regions of interest are stored in a data file . To quantify the purple reporter signal of Zika-positive samples, onboard software calculates the ratio of blue channel values over green channel values, providing similar absorbance-equivalent outputs  and patient samples (diagnostic performance). As the site for the trial, we selected Recife in the Pernambuco State of Brazil, which was the epicentre of the 2016 Zika virus epidemic in Latin America. As a mosquito-borne abrovirus , viral spread in the region was exacerbated by the many peri-urban settings, which, due to standing water, can serve as breeding grounds for mosquitoes such as 25.For this phase of the project, a standardized diagnostic workflow was used in all experiments where, after RNA extraction, samples were tested using Zika virus-specific NASBA and paper-based cell-free reactions Fig. . The PLUWe began the characterization of the diagnostic system by testing the analytical specificity for the American strain of the Zika virus against a panel of seven endemic arboviruses that could, in practice, be found in patients with similar symptoms to those associated with the Zika virus. This included the chikungunya virus (CHIKV) and yellow fever virus (YFV), along with the four serotypes of the dengue virus (DENV-1–4). We also evaluated strain specificity with addition of the African strain of Zika (ZIKV Af). As can be seen by eye, a positive signal (purple colour) was only detected in the presence of the American strain of the Zika virus Fig. , bottom.1 PFU ml−1 , 96.22–99.59 %) and 100%, respectively. Datasets from the field trial diagnostic (0.1603 a.u) and RT–qPCR (Ct ≤ 38) were plotted to show the similar accuracy of the respective thresholds in correctly segregating positive and negative patient samples  in a double-blinded, 65-patient study, with only one false negative, and with the diagnostic sensitivity and specificity of 92.31% and 100%, respectively  and tested for analytical specificity against a panel of eight off-target endemic arboviruses (including the related Mayaro virus (MAYV)). As before, the chikungunya diagnostic test provided 100% analytical specificity for target strains  crisis, the centralized nature of RT–qPCR can limit access to diagnostics and is prone to supply chain disruption during infection surgesStill, important technical challenges remain to be solved ahead of practical implementation. In this report, we have provided validation results for clinical-grade molecular diagnostics using gene-circuit-based sensors and equipment that are low cost and portable. However, as with RT–qPCR, these assays still require liquid handling by skilled users to prepare samples (RNA extraction of 30 samples in about 60 min) and assays. Although we did not experience issues of cross-contamination, the need for manual intervention will increase this risk as efforts move into less technical settings. Continuation of good laboratory practices will be important as technologies make this transition.48. Notably, we also see the implementation of local and distributed reagent manufacturing  as crucial to realizing many of the logistic and economic advantages of distributed diagnostic tools. Work on this is already underway by others in the community, and proof-of-concept efforts show a clear path to ultra-low-cost diagnostic reagents51. Recent proof-of-concept work demonstrated onsite in Chile indicated the cost of producing the cell-free protein expression reaction (5 μl) to be US$0.069, which, here, would replace PURExpress and the related US$2.54 cost . This control serves to guard against false-negative diagnostic results by ensuring that diagnostic samples contain RNA of sufficient quality and quantity. Although we followed gold-standard RT–qPCR methods53. As an early demonstration of gene-circuit-based diagnostics in the field, this work serves to inform the emerging low-cost diagnostics research community of the challenges of taking technologies from the bench and into practice in the field. This transition and the transfer of technology, as we and others have found, hold many challenges55. One main challenge was how to bring high-capacity optical characterization into new environments for distributed testing. We solved this with the development of the PLUM reader, and we anticipate that this hardware solution will help future efforts to validate and implement technologies in low-resource settings. There were also notable logistical challenges in bringing the technology from our laboratory to field sites. Accordingly, we have included brief notes that outline details that we found helpful ) were amplified by PCR using primers (Supplementary Table 15. After PCR-based linearization of the template (New England Biolabs (NEB) Phusion, M0530L or NEB Q5 M0491L) using primers listed in Supplementary Table 6–100 molecules per µl). DNA encoding the target chikungunya sequence was ordered through IDT as dsDNA with a T7 RNAP promoter. The primers used to amplify the target DNA are listed in Supplementary Table DNA encoding the Zika virus target was obtained from the Collins lab in a pET15 backbone7 PFU ml−1) or patient serum samples (140 μl) following the QIAamp Viral RNA Extraction Kit protocol  or by heating samples for 10 min at 95 °C. Processed RNA was then used as input to NASBA isothermal amplification reactions for use in toehold switch reactions.RNA was extracted from lentivirus samples , following the manufacturer’s instructions with slight modifications. In brief, reactions were performed in a 5 μl volume format. Per reaction, 1.67 μl reaction buffer, 0.83 μl of nucleotide mix, 0.05 μl of RNase inhibitors and 500 nM of each primer were mixed along with 1 μl of sample (water containing target RNA or sample lysate). Reactions were assembled at room temperature, incubated at 65 °C for 2 min and then at 41 °C for 10 min, before adding the enzyme mix (1.25 μl). This was followed by incubation at 41 °C for 1 h. Reactions were then added to the PURExpress reaction as previously described at a 1:7 dilution for detection of amplified target(s)15. Reactions were assembled to a final volume of 8 μl. In brief, 40% solution A, 30% solution B, 0.5% v/v RNase inhibitors  and 0.2 µl of 25 mg ml−1 of chlorophenol red-b-D-galactopyranoside  were combined. Linearized DNA encoding the corresponding toehold switch was added to cell-free reactions at 33 nM. For reactions that included isothermally amplified RNA, NASBA products were added at a 1:7 dilution (1.14 μl). The time point data presented  were assembled following the manufacturer’s instructions, as described previouslyed Figs.  and 4a–chttp://n2t.net/addgene:24130; RRID:Addgene_24130). (2) pMD2.G, a VSV-G envelope-expressing plasmid; pMD2.G was a gift from Didier Trono . (3) pMDLg/pRRE, a third-generation lentiviral-packaging plasmid, contains Gag and Pol; pMDLg/pRRE was a gift from Didier Trono . (4) pRSV-Rev, a third-generation lentiviral-packaging plasmid, contains Rev; pRSV-Rev was a gift from Didier Trono .(1) pUltra-hot, third-generation lentiviral vector for bi-cistronic expression of mCherry and the gene of interest; pUltra-hot was a gift from Malcolm Moore .Trigger sequence corresponding to the Zika-virus-specific toehold switches was placed downstream of the mCherry sequence between the P2A and T2A sequences2 in a humidified incubator in DMEM or DMEM supplemented with 4,500 mg l−1 of glucose, ʟ-glutamine and sodium bicarbonate , 10% foetal bovine serum (FBS) and 1% penicillin–streptomycin, approximately, 100 U ml−1 penicillin and 100 µg ml−1 (Gibco).HEK293T cells were grown at 37 °C at 5% CO58. The ratio of plasmid DNA (µg) used was pUltra-hot-T3T8: pMD2.G: pMDLg/pRRE: pRSV-Rev = 4.5: 1.5: 3: 3. Media were then replaced with 10 ml of fresh culture media and incubated at 37 °C. Once mCherry expression was detected, media were collected and centrifuged at 1,000g for 5 min to pellet the debris and the supernatant was syringe filtered using a 0.4 µm filter (Millipore).Once at 70% confluency in a 10 cm plate, cells were transfected with polyethylenimine  by combining a ratio of DNA:PEI at 1:3, following manufacturer’s instructions and incubated overnight at 37 °CLentiX  concentrating solution was added to the filtered supernatant in a 1:3 ml ratio (1 ml of LentiX to 3 ml of virus supernatant), and concentration was performed following manufacturer’s instructions.4 cells per well. Serial dilution of lentivirus aliquot was performed in PBS, with 5–7 technical replicates for each, and 25 µl of the diluted virus was transferred onto the wells containing the cells. The plate was placed at 37 °C for overnight incubation.Virus titration: HEK293T cells were plated at 3 × 1050) to PFU was done using the calculation suggested by the American Type Culture Collection .Plates were analysed for cytopathic effect, and conversion from median tissue culture infectious dose , in Recife, Brazil.AY632535), was isolated from infected mouse brain suspension. All four Dengue virus (DENV (1–4)) serotypes used were isolated from patient samples in Pernambuco State, Brazil: DENV-1, strain PE/97-42735 (GenBank accession no. EU259529), DENV-2, strain PE/95-3808 (GenBank accession no. EU259569), DENV-3, strain PE/02-95016 (GenBank accession no. KC425219) and DENV-4, strain PE/10-0081 (unpublished). Yellow fever virus, strain 17DD (GenBank accession no. DQ100292) is used as a vaccine strain. Chikungunya virus, strain PE2016-480 (unpublished), was isolated from a patient serum in Pernambuco State, Brazil, and chikungunya strain PB302 (unpublished) was isolated from serum of patient in Paraiba State, Brazil. Mayaro virus, strain BR/Sinop/H307/2015 (GenBank accession no. MH513597.1) was provided by the Federal University of Mato Grosso in Sinop, Brazil.Zika virus, American strain PE243 (GenBank accession no. KX197192), was isolated from an infected patient in Pernambuco State, Brazil. Zika virus, African strain MR766 (GenBank accession no. −1 of penicillin (Gibco) at 37 °C under 5% CO2. Mosquito-borne viruses, including ZIKV American lineage, ZIKV African lineage, DENV-1–4, YFV, MAYV, CHIKV PE and CHIKV PB, were titrated using plaque assay with titre ranging from 106 to 107 PFU ml−1. Virus stocks were stored at −80 °C before downstream applications.All viruses used in this study were propagated in Vero cells using DMEM (Gibco) supplemented with 2% of inactivated FBS (Gibco), 2 mM ʟ-glutamine (Gibco), 100 units per ml of streptomycin and 100 μg ml6 PFU ml−1) was extracted using the QIAamp Viral Mini Kit  and eluted in 60 µl of water. After serial dilution of the extracted RNA (from 105 to 10−3 PFU ml−1), samples were assayed in parallel with the gold-standard RT–qPCR and the NASBA/cell-free reactions38. ZIKV American strain (PE243) and CHIKV strain (PE2016-480) were the strains used in those studies.Viral RNA from stock of all viruses  following the manufacturer’s protocols for 10 μl of final volume with primers of both viruses in 800 nM final concentration and probes in 100 nM final concentration. Primers and probes for ZIKV and CHIKV can be found in Table ZIKV and CHIKV RT–qPCR was performed according to protocols established by the CDC, with minor modificationsThe PLUM reader consists of two chambers housed within a laser-cut acrylic box fastened together with metal screws and mounting brackets Fig. . On the The electronic components of PLUM are controlled by a Raspberry Pi 3B. The camera (Raspberry Pi Camera Module V2) in the detection compartment and the LCD display unit (Raspberry Pi Touch Display 7 inch) mounted on the front panel of the PLUM are connected directly to the Raspberry Pi unit with flex cables (Flex Cable for Camera-24 inch/610 mm). The Raspberry Pi controls the detection unit components, with the exception of the incubator, via a 40-pin header ribbon cable connected to the custom-made motherboard PCB (printed circuit board) , vendor.https://github.com/PardeeLab/zikaproject_hardware.All of the information required for the assembly of the PLUM reader is supplied as https://aws.amazon.com/) was used to scale up the storage space of PLUM through a web service interface. GUI-related and workflow-related code can be found at https://github.com/PardeeLab/zikaproject_plumcode.git.A GUI operating on Rasbian was designed using Python 2.7 and open-source APIs . The Amat-test.Statistical analyses for analytical sensitivity experiments were performed on GraphPad Prism 7 (GraphPad Software) using an unpaired https://www.medcalc.org/calc/diagnostic_test.php)48.Final accuracy tests were established using an online mathematical tool provided by MedCalc  in each run. A logistic test was run on data collected for test sensitivity analysis with Zika virus patient samples. According to the time-based thresholds, each patient data point was classified as positive or negative  under protocol 80247417.4.0000.5190 and research ethics board (REB) approval number at the University of Toronto (protocol 39531) and was conducted in accordance with relevant regulations and guidelines, including the ethical principles for medical research involving human subjects designed by the World Medical Association Declaration of Helsinki. Patient samples were obtained from suspected arbovirus infections, from patients who presented arthralgia, fever, exanthema and other related symptoms in an endemic area of several arboviruses in Latin America. Informed consent of all individuals included in this study was waived by the FIOCRUZ-PE IRB for diagnostic specimens.Further information on research design is available in the Supplementary InformationSupplementary methods, figures, tables and references.Reporting SummarySupplementary DataNucleic acid sequences.


Monoclonal antibodies (mAbs) have proven effective for the treatment of ebolavirus infection in humans, with two mAb-based drugs Inmazeb™ and Ebanga™ receiving FDA approval in 2020. While these drugs represent a major advance in the field of filoviral therapeutics, they are composed of antibodies with single-species specificity for Zaire ebolavirus. The Ebolavirus genus includes five additional species, two of which, Bundibugyo ebolavirus and Sudan ebolavirus, have caused severe disease and significant outbreaks in the past. There are several recently identified broadly neutralizing ebolavirus antibodies, including some in the clinical development pipeline, that have demonstrated broad protection in preclinical studies. In this review, we describe how structural biology has illuminated the molecular basis of broad ebolavirus neutralization, including details of common antigenic sites of vulnerability on the glycoprotein surface. We begin with a discussion outlining the history of monoclonal antibody therapeutics for ebolaviruses, with an emphasis on how structural biology has contributed to these efforts. Next, we highlight key structural studies that have advanced our understanding of ebolavirus glycoprotein structures and mechanisms of antibody-mediated neutralization. Finally, we offer examples of how structural biology has contributed to advances in anti-viral medicines and discuss what opportunities the future holds, including rationally designed next-generation therapeutics with increased potency, breadth, and specificity against ebolaviruses. Ebolaviruses are the genus from the family of Filoviridae that includes six distinct viral species: Zaire ebolavirus [represented by Ebola virus (EBOV)], Bundibugyo ebolavirus [(Bundibugyo virus (BDBV)], Sudan ebolavirus [Sudan virus (SUDV)], Taï Forest ebolavirus [Taï Forest virus (TAFV)], Bombali ebolavirus [Bombali virus (BOMV)], and Reston ebolavirus [Reston virus (RESTV)]. Of these six species, EBOV, BDBV, and SUDV cause the most severe disease in humans. Together with the related Marburg virus representative of genus Marburgvirus, filoviruses have caused at least 30 major, deadly outbreaks since their initial discovery in 1967, with increasing frequency and severity in last decade . Two therapeutic interventions currently have FDA approval, both of which consist of monoclonal antibodies (mAbs) and are only effective against EBOV. The single mAb therapeutic named Ebanga™ includes Ansuvimab-zykl . Ebanga™Despite the success of mAb therapeutics for the treatment of autoimmunity and cancer, mAb therapeutics for combatting pathogens have been slow to develop. Other than Ebanga™ and Inmazeb™, there exists only two other antiviral antibody therapeutics with full FDA approval , as wel MINFLUX , 134, arCM wrote the manuscript and generated the figures. PG, JC, and AW helped write and edit the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by the NIH (U19 AI142785 and U19 AI109711).JC has served as a consultant for Luna Biologics and Merck Sharp & Dohme Corp., is a member of the Scientific Advisory Board of Meissa Vaccines and is Founder of IDBiologics. The Crowe laboratory at Vanderbilt University Medical Center has received sponsored research agreements from Takeda Vaccines, IDBiologics and AstraZeneca.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.


Methanoculleus bourgensis E02.3, a methanogenic archaeon belonging to the Methanomicrobiales, both isolated from a commercial biogas plant in Germany. The virus consists of an icosahedral head 60 nm in diameter and a long non-contractile tail of 125 nm in length, which is consistent with the new isolate belonging to the Siphoviridae family. Electron microscopy revealed that Blf4 attaches to the vegetative cells of M. bourgensis E02.3 as well as to cellular appendages. Apart from M. bourgensis E02.3, none of the tested Methanoculleus strains were lysed by Blf4, indicating a narrow host range. The complete 37 kb dsDNA genome of Blf4 contains 63 open reading frames (ORFs), all organized in the same transcriptional direction. For most of the ORFs, potential functions were predicted. In addition, the genome of the host M. bourgensis E02.3 was sequenced and assembled, resulting in a 2.6 Mbp draft genome consisting of nine contigs. All genes required for a hydrogenotrophic lifestyle were predicted. A CRISPR/Cas system (type I-U) was identified with six spacers directed against Blf4, indicating that this defense system might not be very efficient in fending off invading Blf4 virus.Today, the number of known viruses infecting methanogenic archaea is limited. Here, we report on a novel lytic virus, designated Blf4, and its host strain  These methanogenic archaea (methanogens) thrive by coupling the conversion of simple C1 and C2 compounds  systems, prokaryotes often have clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, which are the adaptive prokaryotic immunity. They are present in most sequenced archaeal genomes (>90%). Today, the CRISPR/Cas systems are divided into 2 classes, 6 types, and >30 subtypes ,16. The in situ [Methanothermobacter marburgensis-infecting viruses ψM1 and ψM2, and the Methanothermobacter wolfeii provirus ψM100 [Methanobacterium [Methanococcus voltae, virus(-like) particles were reported but not characterized in detail [Methanosarcina sp. (MetMV) was predicted via a metagenomic approach [Whether CRISPR/Cas systems play a significant role in fending off viral infections of methanogenic archaea involved in anaerobic digestion is unclear. The emerging evidence for the relevance of viruses to the structure and population dynamics of archaeal communities is still largely based on observations in situ ,20,21. Mus ψM100 ,23,24. Facterium ,26. For n detail ,28. Recen detail . A seconapproach . For theapproach ,31.Methanoculleus bourgensis E02.3 and its lytic virus, Blf4, from a commercial biogas plant in Germany. The virus belongs to the Siphoviridae, and is the first one described to infect a member of the Methanomicrobiales, expanding the repertoire of isolated viruses infecting methanogens.In this study, we isolated 2/CO2/H2 . “Modified Basal Medium” (MBM) and solid “Modified Basal Agar” (MBA) were used to isolate and culture methanogenic archaea present in sludge obtained in April 2013 from a full-scale commercial anaerobic digester in Germany (designated “BG1” in [2/CO2 (80:20 (v/v), 1.5 × 105 Pa) for 12 days. In this initial isolation step, the media contained 100 µg mL−1  of streptomycin and ampicillin each in order to inhibit bacterial growth. Obtaining a pure culture from a single colony on MBA and assessment of purity by phase-contrast and epifluorescence microscopy was conducted as described [578) or 600 nm (OD600) at 37 °C, 40 °C, 45 °C, and 50 °C. Growth onsodium formate  at 45 °C was assessed as described [All manipulations were made under anaerobic conditions, either by applying standard anaerobic techniques  or by wo“BG1” in ). MBM an“BG1” in , withoutescribed . Growth escribed . MethanoM. bourgensis E02.3, as well as its phylogeny, was assessed by analyzing a nearly complete fragment (1395 bp) of the 16S rRNA gene sequence, amplified by PCR. A phylogenetic tree was constructed using the neighbor-joining method and the Jukes–Cantor distance correction with MEGA based on a Geneious10.2.5 CLUSTAL W alignment of sequences representing the genus [M. bourgensis E02.3 was sequenced. Chromosomal DNA was isolated from M. bourgensis E02.3 using the Wizard Genomic DNA Purification Kit  as recommended by the manufacturer. Illumina shotgun paired-end sequencing libraries were prepared using the Nextera DNA Flex Library Preparation Kit  according to the manufacturer’s instructions. The sample was sequenced on the NextSeq500 using the NextSeq 500/550 Mid Output Kit v2.5 sequencing chemistry (300 cycles). Additionally, 400 ng of high-molecular-weight genomic DNA were sequenced using the SQK-RBK004 rapid sequencing kit, FLO-MINI106 flow cells, and a MinIon device (MinKNOWversion 20.06.4) . Of the 2,213,275 paired-end reads, 15% overlapped and were merged using bbmerge [https://blast.ncbi.nlm.nih.gov/Blast.cgi; accessed on 8 March 2021); CRISPR/Cas systems were identified using CRISPRFinder [fla-genes [M. bourgensis E02.3 is available at GenBank under the accession number GCA_018495055.1.The taxonomic rank of he genus  and boothe genus . Additio bbmerge . A hybri bbmerge , and operagment 15 bp of t bbmerge , respectPRFinder , MacSyFiPRFinder , and CRIPRFinder ) and embla-genes . Protospla-genes ,47. The g for 10 min, the supernatant was filtered through a 0.45 µm membrane. M. bourgensis E02.3 was infected with the filtrate in a standard double-layer plaque assay analogous to a procedure described previously [578 of approximately 0.1) growing strain E02.3, combined with 2.5 mL of semi-solid molten MBA (0.7% (w/v) agar), and poured on fresh MBA plates. The plates were incubated at 45 °C with H2/CO2 for 14 days. Single plaques were harvested, serially diluted in MBM, and the lytic virus was again isolated via the plaque assays to ensure that a pure strain was obtained. For amplification of virus biomass, the volumes of the Blf4 lysate (up to 8 mL) were increased to lyse increasing volumes (up to 160 mL) of M. bourgensis E02.3 cultures incubated with slight agitation at 45 °C over several infection/lysis cycles. For subsequent analyses, the cultures were cleared by centrifugation at 10,000× g for 30 min after complete cell lysis (24–48 h) had occurred, and aliquots of the supernatants were either stored anaerobically in 5% (v/v) glycerol at −80 °C (Blf4 virus stock), subjected to isolation of the virus (see below), or used to assess the host spectrum of the Blf4 virus. For the latter, three Methanoculleus species, M. bourgensis MS2T , M. marisnigri AN8 (DSM 4552), and M. thermophilus CR1T  , were cultivated in the respective media suggested by DSMZ  with H2/CO2 (80:20) and supplemented daily, as the energy substrate. For lysis assays, strains were grown in 5 or 50 mL culture to an optical density of 0.15–0.2 and supplemented with 0.2–0.5 mL of virus lysate passed through a 0.2 µm sterile filter . The optical density was followed until complete lysis occurred or the stationary phase was reached.Sludge from the anaerobic digester was diluted 1:4 with MBM and manually homogenized. After centrifugation at 3000× eviously . BrieflyM. bourgensis E02.3 was applied to either pioloform- or formvar-carbon-coated, 300- or 400-mesh copper grids  stained with 1–2% aqueous uranyl acetate solution and examined by TEM  at an accelerated voltage of 80 kV.The Blf4 virus was visualized by transmission electron microscopy (TEM) using conventional negative staining. To this end, virus stock suspension or Blf4-infected v/v), incubated at 4 °C for 16 h, and centrifuged at 3000× g for 20 min. The supernatant was passed through a 0.45 µm pore filter  and subjected to ultracentrifugation . The sediment was resuspended and, after another ultracentrifugation under the same condition, further purified by ultrafiltration through a 100 kDa molecular weight cut-off microcentrifuge device  at 1000× g. The retentate was treated with DNaseI (Thermo Fisher Scientific) to remove host DNA.Lysate (80 mL) containing Blf4 virus was thoroughly mixed with chloroform at a final concentration of 5%  as per the manufacturer’s instructions. Illumina short-read shotgun sequencing of virus DNA from Blf4 with standard NGS library preparation, followed by paired-end 300 bp sequencing on a MiSeq system (Illumina) was conducted at the Genome Center of the Technische Universität Dresden. The sequencing run generated 7,813,662 paired-end raw reads. Filtering and subsampling were conducted as described . Assemblak point . Annotatak point . Furtherak point , and theak point . Virus cak point ,54. The 420 autofluorescence  or formate (150 mM) for growth. Growth with methanol (125 mM) or acetate (120 mM) as sole energy sources was not observed. Growth of the E02.3 strain required acetate (10–40 mM gave no phenotypic difference) to be present in the medium. Growth was fastest at 40 °C and 45 °C   and a BLASTN analysis of the M. bourgensis E02.3 draft genome. The genes encoding formate dehydrogenase  were found in contig 4. Genes for  enzymes involved in the assimilation of acetate were found scattered across the genome—putative acetate-CoA ligase (BN140_034) in contig 2, acetate kinase (BN140_1312) in contig 4, phosphotransacetylase (BN140_0884) in contig 3, acetyl-CoA-synthetase (BN140_2190) in contigs 8 and 5, and carbon monoxide dehydrogenase/acetyl-CoA synthase complex (MMAB1_3162) in contig 4.Comparative analysis of the nearly complete 16S rRNA gene (1395 bp) from the E02.3 isolate with other sequences in GenBank (BLASTN) showed that this strain belongs to the genus B065298) . Both achenotype  and are,and MS2T . Based oflaJ, flaI, flaH, flaF, flaG, and the flagellin flaB) in contig 1. The deduced proteins were 48.1 to 89.5% identical to those from M. thermophilus and M. marisnigri. A CRISPR/Cas system type I-U was identified in contig 7  .One locus encoding the proteins for the archaeal flagellum was identified  a–c. The M. bourgensis E02.3 array, in which only two spacers had one mismatch (spacers 13 and 17), three spacers had two mismatches , and one had five mismatches (spacer 28). In comparison, M. bourgensis MS2T and MAB1 had 16 spacers against the Blf4 virus (two without mismatches). No spacers without a mismatch were present in the array of M. bourgensis E02.3 and only two spacers completely identical to Blf4 were present in the arrays of M. bourgensis MAB1 and MS2T, respectively. It has been shown that a 100% match is not necessary for the functionality of CRISPR/Cas systems [T, and MAB1 had only one spacer in common  using BLASTN. Thus, spacers with mismatches were also analyzed. Six spacers against the Blf4 virus were identified in the  systems ,57. Notaredicted d using tand MAB1 e,f.M. bourgensis E02.3. The draft genome was compared with the known restriction–modification systems from M. bourgensis MS2T and MAB1 (using BLASTN). Genes were identified, which are annotated in M. bourgensis MS2T and MAB1 as enzymes of the type III restriction–modification system in contig 3 (nt: 259043–263631). A further section in contig 3 is very similar to another type III restriction–modification of M. bourgensis MS2T (BN140_0641/0642) and MAB1 (MMAB1_0829/0830), but an intermediate section of 1251 bp  differs significantly in the primary structure. Surprisingly, none of the known type I restriction–modification systems from M. bourgensis MS2T  or MAB1 (MMAB1_2046-2048) were identified.As for further mechanisms of defense against foreign DNA, restriction–modification systems were searched in the genome sequence of M. bourgensis E02.3 24 h after they were challenged with the virus  [Siphoviridae. The portal proteins are involved in virus replication (virion assembly), DNA packing, and DNA delivery [S-adenosyl-dependent) methyltransferases, potential antiCRISPR proteins), and for the maturation of the virus  and the products of ORFs 62 and 63 are putative endonucleases. Further genome analysis using VIRFAM for a head–neck–tail module search supported the classification as Siphoviridae type 1 (no cluster assigned), as well as protein function prediction for the major capsid protein (MCP)(ORF14), the portal protein (ORF11), the terminase (ORF7), the head–tail adapter protein (ORF16), the head closure protein (ORF17), the neck protein (ORF19), and the tail completion protein (ORF20) , Methanothermobacter wolfeii prophage ψM100 (taxid 173824), Methanobacterium phage ψM2 (taxid 77048), Methanobacterium formicicum virus Drs3 (taxid 1430441), Methanosarcina-infecting virus MetSV (taxid 2035535), and Halorubrum phage CGphi46 (taxid 75406). Furthermore, VIRFAM (head–neck–tail module) analysis of these four Siphoviridae members infecting archaea and Blf4 showed, beside the different genome sizes, different arrangements of the genes for the structural proteins . This length is comparable to other members of the for BJ1) . The GC delivery . ORFs 12F20) see . No signproteins .M. bourgensis E02.3, were isolated from a commercial biogas plant operated at approximately 40 °C [Methanoculleus. The high degree of protein similarity that was deduced from the genomic sequence to other M. bourgensis strains (MS2T or MAB1), for example, those involved in energy metabolism (hydrogenotrophic methanogenesis), supports this notion.The Blf4 virus described here is only one of the few known to infect methanogenic archaea. Blf4 virus and its host, ly 40 °C . The groSiphoviridae virus family. Viruses of this family are known to infect bacteria or archaea. The morphology of Blf4 resembles that of the methanogenic viruses Drs3, ψM1, and ΦF3 [Siphoviridae is a circular, permuted, terminally redundant genome that is packaged by a “headful mechanism” as linear dsDNA into the viral particle, and circularizing after injection into the host [Escherichia coli phage HK97 .Pseudomonas, Pectobacterium, Listeria, Streptococcus, Moraxella, or Sulfolobus [M. bourgensis E02.3, despite the presence of a CRISPR/Cas system primed against it. The function of putative Blf4 Acrs and their potential interaction with the host’s CRISPR/Cas system is unclear, but it is expected to differ from the SIRV2 Acrs [Sulfolobus contains types I-A, I-D, and III-B; M. bourgensis E02.3 contains type I-U), and (b) no similarity between the respective Acrs could be detected. Possible explanations for the very narrow host range of Blf4 observed in this study might be the fact that M. bourgensis E02.3 lacks type I restriction–modification systems, which is encoded by other M. bourgensis , or the presence of more and different spacers against the virus in the CRISPR array of the tested strains.In addition to proteins for virus propagation and structure, Blf4 also codes for proteins that may be involved in host defense inhibition, namely antiCRISPR (Acr) proteins, which are able to interfere with the host’s CRISPR/Cas system defenses (reviewed in ). Only alfolobus ,71,72,73RV2 Acrs , as (a) M. bourgensis E02.3′s cell surface and its appendages , which are rather distinct from those of M. bourgensis E02.3. Both strains were not lysed by Blf4. In contrast, M. bourgensis MS2T, when challenged with Blf4, was also not lysed, despite encoding a flagellum very similar to that of M. bourgensis E02.3. The observed immunity against Blf4 in the Methanoculleus strains examined here might be based on a combination of different mechanisms, such as a defense system (CRISPR/Cas or a restriction–modification system in the case of M. bourgensis MS2T) and incompatible receptors (in the case of M. thermophilus or M. marisnigri).The finding that Blf4 attaches to both pendages  suggestsMethanosarcina and Methanoculleus [Methanoculleus genus might exert a profound effect on their host’s abundance and, thus, on the efficiency and the economy of the whole biogas process. Since only a few viruses of methanogens are known, their overall impact on methanogenic activity can only be guessed. Therefore, exploring the genetic diversity of viruses infecting methanogens and the dynamics of virus–host interactions will aid our understanding of anaerobic digestion and of anaerobic systems in general.Using culture-independent sequencing approaches, a number of methanogenic archaeal taxonomic groups have been identified as being potentially relevant for the biogas process. In biogas plants operated in a similar fashion to the one from which Blf4 originated, these abundant methanogens included the genera oculleus . In factoculleus . Assumin