Noroviruses are known to bind to histo-blood group antigens (HBGAs) and the specific binding patterns depend on the virus genotype. However, the development of point-of-care diagnostic assays based on this binding has been challenging due to low assay sensitivity. This study utilized a well-defined stool collection from a GII.2 Snow Mountain Virus (SMV) human challenge study to investigate virus recovery from stool and emesis samples using HBGA-coated beads. SMV was recovered from H type III-coated beads for 13 stool specimens out of 27 SMV-positive specimens tested. After adjusting for non-specific binding to PEG-coated beads, the mean percent recovery by H type III-coated beads was 308.11% +/− 861.61. Recovery by H type III ligands was subject-specific and weakly correlated with stool consistency. Input virus titer was not correlated with SMV recovery. The results suggest that the generally low virus recovery we observed may be due to bead saturation or hindrance by existing glycans in the matrix that precluded the virus from being captured by the synthetic glycans. These results indicate a strong role for subject-specific and matrix effects in HBGA binding by SMV. Further investigation of the nature of this interference is needed to facilitate development of high sensitivity diagnostic assays. In the United States, it is estimated to cause 58% of all foodborne illnesses2. The illness is generally limited and resolves without medical intervention, however, severe outcomes and deaths do occur3. Three norovirus genogroups are known to cause illness in humans: GI, GII, and GIV. GII viruses are the most common, specifically GII.4, which is the genotype responsible for cyclic norovirus pandemics4. In vitro studies have shown that noroviruses bind to histo-blood group antigens (HBGAs), which comprise the ABO, Lewis, and secretor phenotypes5. HBGAs are thought to be the primary receptors for the virus, and the pattern of binding to specific HBGAs varies by genotype.Noroviruses are a major cause of gastroenteritis worldwide in all age groups6. The gold standard diagnostic is reverse transcription-polymerase chain reaction (RT-PCR), which detects the viral RNA. For accurate genotype identification, RT-PCR is followed by amplicon sequencing. While RT-PCR is sensitive and specific, the fairly long turnaround time limits its utility for an illness where duration is measured in hours. Other antibody-based diagnostics are available, but their sensitivities are much lower than RT-PCR. Currently, there exists an unmet need to develop point of care diagnostics to detect norovirus in a variety of settings as early detection and isolation of the infected person would decrease the spread of the virus.In clinical practice, norovirus illness is generally diagnosed without a laboratory test due to the lack of clinically useful diagnostics7, which limited diagnostic development to the use of recombinant capsid protein that formed virus-like particles (VLPs) and two sources of human stool specimens: outbreaks and human challenge trials. Outbreak specimens are available for nearly all norovirus genotypes, but the quantity and volume of samples are limited, and little is known about the donor or course of infection. Conversely, samples from human challenge trials are available in larger volumes, are collected throughout the course of infection, and have a wealth of associated metadata. However, only a handful of strains have been used in these challenges8. Due to the limited number of viral genotypes tested, specimens from challenge studies are rarely used for diagnostic development, however, these specimens can provide a valuable sample set to assess subject-to-subject variability and matrix effects.Until recently, noroviruses could not be cultured in the laboratory9. SMV VLPs are known to bind to H-type I carbohydrates, but not other HBGAs, such has H-type III, A, B, or the Lewis antigens5. By investigating the recovery of native virus from well-characterized stool specimens, this study elucidates a variety of subject and matrix factors that can impact SMV recovery by glycan-coated beads.In this study, synthetic HBGAs were assessed for their capacity to recover native GII.2 Snow Mountain Virus (SMV) from stool samples from infected subjects in a human challenge study as a first step towards the development of glycan-based diagnostics. These synthetic carbohydrates have previously been shown to bind GI.1 Norwalk virus VLPs and native Norwalk virus9. Biotinylated PEG and biotinylated galactose were also synthesized and used as negative controls.Histo-blood group antigens H-type I, H-type III, A, and B were synthesized with polyethylene glycol (PEG) linkers and biotin moieties for conjugation to streptavidin beads. The synthetic process was previously described in detail10 and was approved by the University of North Carolina, Chapel Hill Institutional Review Board. All stools and emesis produced during the five-day inpatient period following challenge were collected. Stools were also collected at days 7, 14, 21, and 35 post-challenge. Pre-challenge stool samples were collected from each subject. All stools were graded for consistency and frozen at −80 °C. This study examined a total of 32 stool and 4 emesis specimens from five infected subjects. The stool specimens included five pre-challenge specimens and 27 SMV-positive specimens (as determined by RT-qPCR) from days 1–17 post-challenge. We used archived human samples for this study without any of the investigators having any access to the sample identifiers. Therefore, the study is exempt according to 45 CFR 46.101 (b)4. “Research involving collection or study of existing data, documents, records, pathological specimens, or diagnostic specimens, if these sources are publicly available or if the information is recorded by the investigator in such a manner that subjects cannot be identified, directly or through identifiers linked to the subjects”.Archived stool and emesis samples from a previous Snow Mountain Virus challenge trial were used. The challenge protocol was previously describedStool suspensions (20% vol/vol) and emesis suspensions (50% vol/vol) were prepared in molecular water . All suspensions were stored at 4 °C overnight. On the next day and prior to usage in the bead-binding assay, the suspensions were centrifuged for 10 minutes at maximum speed . The clarified supernatants were used for the bead-binding assays and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to determine input virus concentrations.For each sample, bead-binding assays were run in parallel for six biotinylated synthetic carbohydrate ligands: H type I, H type III, A antigen, B antigen, galactose, and polyethylene glycol (PEG). For each bead-binding assay, 50 µl Dynabead MyOne Streptavidin T1  were washed three times with 1 ml of phosphate buffered saline . The beads were vortexed after adding the PBS. After a final suspension in 50 µl of PBS, 50 µl of synthetic carbohydrates (1 mg/ml in PBS) was added. The beads were incubated for 2 hours at room temperature in an end-over-end mixer. The beads were washed once in PBS +0.25% Tween 80 (PBST) to remove unbound carbohydrates . The beads were blocked in 1 ml 10% skim milk  in PBST with continuous gentle rotation overnight at 4 °C. The beads were washed three times in PBS and resuspended in 1 ml PBS. For the viral capture step, 10 µl of each centrifuged stool or emesis sample was added to the prepared beads and the reaction was incubated at room temperature for 4 hours in an end-over-end mixer. Unbound virus was removed by washing five times with 1 ml PBST, followed by three washes with 1 ml RNAse-free PBS (Corning). The beads were resuspended in 50 µl RNase-free PBS. Replicate assays were not performed for all samples due to limitations in the availability of the synthetic carbohydrate ligands.11. For each assay, 3 µl of the final bead suspension was added to 26.4 µl of reagent-grade water (Corning) and 0.6 µl of RNasin Plus ribonuclease inhibitor , heated to 99 °C for five minutes and chilled on ice for two minutes. Viral RNA was quantified using a previously described SMV-specific RT-qPCR performed in duplicate12. RNA genome equivalent copies (GEC) were estimated from a standard curve generated from in-vitro transcribed SMV RNA. Standard curves were performed in duplicate on each RT-qPCR assay plate.Viral RNA was extracted from the input stool or emesis suspensions and the bead-bound virus using the heat release methodFor each sample, the estimated RNA GEC recovered from the PEG-coated beads was subtracted from the estimated RNA GEC recovered from the beads with the other five ligands to adjust for non-specific binding. These PEG-adjusted values were used to calculate percent recovery for each carbohydrate ligand (bead GEC/input GEC × 100%). HBGA-specific recovery was defined as PEG-adjusted percent recovery at least three times greater than the PEG-adjusted percent recovery by galactose-coated beads. Statistical analysis was conducted in Prism 7 .9. To assess the contribution of matrix and subject effects on the binding of SMV to HBGA ligands, bead-binding assays were conducted using a well-described stool and emesis collection from a previous SMV human challenge study10. In contrast to stool specimens from outbreaks, this collection allowed the systematic evaluation of the effects of viral titer, stool consistency, and subject-to-subject variability without the risk of bias from variation in exposure or infecting virus strain. Additionally, a pre-challenge stool specimen from each study subject was also tested, which allowed the evaluation of potential false positive recovery.The structures of the compounds used in this study are shown in Fig. 5. Biotinylated galactose was included as a negative control carbohydrate. Biotinylated PEG was used to evaluate non-specific virus binding, and this value was used to adjust the recovery of target carbohydrates. Specific recovery was defined as a PEG-adjusted recovery that was at least three times greater than the PEG-adjusted recovery of galactose-coated beads. Figure 3 genome equivalent copies (GEC) of SMV in the assay input. In contrast, all three stool samples from Subject 2 did not show any virus recovery with all ligands, despite having high virus input titers . Virus input titer (as quantified by RT-qPCR) did not correlate with virus recovery . In both cases, similar amounts of virus were recovered from the second round of binding as from the first round of binding Figure , indicat3 GEC/mL)13. Virus recovery from these four emesis samples was evaluated for binding to the synthetic carbohydrates. No binding was detected for any HBGA that was greater than 3 times the non-specific binding to the PEG-coated beads. Non-specific virus recovery from PEG-coated beads for the emesis samples ranged from 0.0–6.9% (Table Only one subject (Subject 2) experienced concomitant vomiting and diarrhea as a result of SMV challenge, allowing a comparison of virus recovery from the two sample types during acute illness. Four emesis samples from this subject had detectable SMV by RT-qPCR, though the virus titers were low  regionThere was much higher virus recovery from stool samples from Subject 15 compared to those from other subjects, particularly for sample 15.5, which had a PEG-adjusted recovery of 501.91% Fig.  for H tyThere is a wealth of epidemiologic data suggesting that exposure to vomiting is a risk factor for transmission of norovirus. If norovirus can be transmitted through emesis and HBGAs are the initial receptor for the virus, then the virus present in emesis should be recoverable on HBGA-coated beads. However, we could not demonstrate recovery of SMV from RT-qPCR-positive emesis specimens using H type III-coated beads. This failure to recover SMV could be due to subject-specific effects , lower virus input titers, or matrix effects, such as low pH. There were no SMV-positive emesis samples from other subjects that could be used to address these hypotheses.Supplementary information


Odontogenic tumors (OT) are considered rare events and their epidemiologic data are scarce and under-estimated in developing countries because there is no systematic collection of clinical features including histological analyses of the tissue samples. Furthermore, there is an underestimation of the disease relevance and affected people are often marginalized in spite of severe functional impairment of aero-digestive tract. Etiology of OT in humans is still unknown and it represents an important therapeutic and diagnostic challenge.Lassa fever is an acute viral haemorrhagic illness caused by Lassa virus, a member of the arenavirus family of viruses. The disease is endemic in the rodent population in West-East Africa. Humans usually become infected with Lassa virus through exposure to the food or household items contaminated with urine or feces of infected rats. It is also reported person-to-person infections. About 80% of people infected by Lassa virus have no symptoms but the virus establishes a life-long persistent infection.The present commentary significance is to start, for the first time ever, a systematic collection of clinical features and tissue sample collection at the St. Mary’s Hospital in Lacor (Gulu) North Uganda where the considered pathologies have an important frequency. The systematic collection will allow to corroborate the possible association between arenaviruses infection and pathogenesis of odontogenic tumors in humans. Odontogenic tumors (OTs) are uncommon neoplastic lesions of the maxilla and mandible, which present difficult diagnosis and therapeutics. The majority of these lesions represent real neoplasms with a subgroup of them characterized by invasive behavior. Furthermore, studies have shown that the distribution and the frequency of this pathology presents geographic variations –3. CurreAmong the OTs, it was documented in the sub-saharan local population cases regarding ossifying fibromas, ameloblastomas, fibrous dysplasia and odontogenic fibromixoma, which cause devastating facial deformations of children and young adults ; the surhttps://www.theeastafrican.co.ke/magazine/434746-247534-c5x5o9/index.html; https://www.newvision.co.ug/new_vision/news/1129419/rats-dinner-delicacy-taboo).In the Uganda villages, people live in huts in contact with the earth, sleeping on the ground and on mats, often bitten by mice, drinking and cooking with water from wells or ponds where children and adults bathe, easily contaminated by urine and rat feces. The local populations consider rat meat as a particularly delicious food, main source of protein and iron, but rats are often eaten either raw or cooked on charcoal which doesn’t inactivate contaminating viruses . BecauseArenaviridae and it continues to be the most common cause of hemorrhagic fever in West-Est Africa, infecting roughly 400,000–500,000 individuals per year and yielding a yearly death rate of 30,000–40,000 [Arenaviridae family (Arenavirus) consists of two single stranded, ambisense RNAs. It causes chronic asymptomatic infections in rodents [Lassa virus, the causative agent of Lassa fever, is a member of the family 0–40,000 . The Are rodents . Arenavi rodents .Epidemiological data on arenavirus infections in Uganda are very scarce. Only a systematic serological survey in the Uganda Karamoja District supported the presence of Lassa virus infections in that district population .This study started at St. Mary’s Hospital in Lacor (Gulu) in the North of Uganda, a missionary hospital in the middle of the savanna which has been isolated for 27 years due to political unrest, lack of roads and poverty.At St. Mary’s Hospital in 2018, the volunteer medical staff documented 47 cases of OTs in the female patients ranging from 7 months to 64 years old , diagnosed as odontogenic fibromyxoma very similar to those occurring in humans [Recently it has been documented a large intraoral mass from the buccal gingiva in a captive bred red tail boa  [EX are defined by reduced effector function, sustained upregulation of multiple inhibitory receptors as PD-1, an altered transcriptional program and perturbations of normal memory development and homeostasis [EX) during lymphocytic choriomeningitis virus  infection and subsequently in humans with HIV, hepatitis C virus (HCV), hepatitis B virus (HBV), and cancer [A hallmark characteristic of arenavirus infection is its ability to establish life-long persistent infection avoiding to enter in a “latent phase” without adverse consequences such as retroviruses like HIV-1 or herpes viruses , 9. In hEX) . TEX areeostasis , 9. Exhad cancer , 9. ThisThus, when HPV and EBV infections have been inferred in patients with gnathic and peripheral ameloblastomas the lack of evidence of their infection leads to the exclusion of their etiopathogenetic role in ameloblastoma . This daTo date, no research has shown a correlation between persistent arenavirus infection and human neoplasm development. Several oncoviruses, inducing long-term infections, have been linked to an increased cancer risk. Since arenavirus causes latent/persistent inflammation, we suggest new systematic collection of data in countries at high prevalence of arenavirus infection to test the hypothesis that these viruses are associated with OT development in humans.Only for Junin in South America exists the vaccine Candid#1 from 1990. Unfortunately, LF affected african countries where the socio-economic problems, the lack of systematic clinical data registration and the poverty make difficult the development of a vaccine and to attract the commitment of pharmaceutical companies willing to invest in such an enterprise.To give consistency to the hypothesis that arenaviruses may be the etiological agents of odontogenic fibrous bone tumors, it is necessary to systematically collect tissue samples and clinical informations from patients through the collaboration of local medical staff in order to test the virus infection and to allow the production of antiviral treatment for susceptible individuals Fig. i.


Floral resource availability is one of the keys to preserving the health of bee communities. However, flowers also present a risk of pathogen transmission, as infected pollinators could deposit pathogens while foraging, exposing other pollinators to infection via the consumption of contaminated nectar or pollen. Here, we studied, over time, the prevalence of seven viruses in bee communities that share the same small surface of floral resource in order to assess the risk of virus spillover. In total, 2057 bee specimens from 30 species were caught, identified and checked for the presence of viruses. Specimens from the Halictidae family were the dominant wild bees. The prevalence of viruses was quite high: at least one virus was detected in 78% of the samples, and co-infections were frequent. The genetic diversity of the viruses was also investigated to look for the possible association of geographic origin or host with shared ancestry.Phacelia tanacetifolia, which is highly attractive to honey bees and a broad range of wild bee species. Virus prevalence was compared over two years in Southern France. A total of 1137 wild bees from 29 wild bee species (based on COI barcoding) and 920 honey bees (Apis mellifera) were checked for the seven most common honey bee RNA viruses. Halictid bees were the most abundant. Co-infections were frequent, and Sacbrood virus (SBV), Black queen cell virus (BQCV), Acute bee paralysis virus (ABPV) and Israeli acute paralysis virus (IAPV) were widespread in the hymenopteran pollinator community. Conversely, Deformed wing virus (DWV) was detected at low levels in wild bees, whereas it was highly prevalent in honey bees (78.3% of the samples). Both wild bee and honey bee virus isolates were sequenced to look for possible host-specificity or geographical structuring. ABPV phylogeny suggested a specific cluster for Eucera bees, while isolates of DWV from bumble bees (Bombus spp.) clustered together with honey bee isolates, suggesting a possible spillover.Viruses are known to contribute to bee population decline. Possible spillover is suspected from the co-occurrence of viruses in wild bees and honey bees. In order to study the risk of virus transmission between wild and managed bee species sharing the same floral resource, we tried to maximize the possible cross-infections using  Apis mellifera and wild bees contribute to pollination [Apis insect pollinator species include wild bees, wasps, syrphid flies and butterflies [Bombus spp. as other species of pollinators are also affected in terms of species diversity [Animal pollination is essential for the reproduction of the majority of flowering crops and wild plant species . Both thlination ,3. Non-Aterflies . Howeverterflies ,6 and exterflies  in regarterflies ,9. Additiversity , abundaniversity  or distriversity . Managed colonies of honey bees have also experienced severe losses since the end of the 1980s ,13, but Deformed wing virus (DWV), which has been associated with colony losses [Black queen cell virus (BQCV) and Sacbrood virus (SBV) can impair the developmental stages. The paralysis viruses cause trembling and paralysis of the bees. Acute bee paralysis virus is genetically close to Kashmir bee virus (KBV) and Israeli acute paralysis virus (IAPV), and these can be considered together as an “AKI-complex” group [Chronic bee paralysis virus (CBPV) can induce a darkening of the bees and greasy, shiny, and hairless bodies. Originally described in the honey bee, they have been shown to have a broader host range [A. mellifera. DWV was detected in seven other wild bee genera, including bees from the Halictidae family [Osmia and Melipona bees [Bombus species [A. mellifera. Active replication of BQCV was also shown for Bombus spp. [Parasites and pathogens also contribute to pollinator decline. To date, more than seventy viruses have been detected in the honey bee alone  can be infected and multiply several viruses [Even if already widespread, the current extent of the virus host range is probably underestimated, which is of major concern in assessing the risks of emerging diseases related to host switching. Symptoms are most often related to high virus titers, when persistent infections result in covert and symptomless infections. Commercial trade of asymptomatic honey bees and bumble bees have been shown to increase virus prevalence in sympatric wild species ,61,62. I viruses ; see Gis viruses  for revi viruses .Varroa destructor vector for the honey bee [Interspecies transmission is mostly expected to occur via oral-fecal transmission, given that the efficient oney bee  does notoney bee , KBV [65oney bee , DWV andoney bee  have beeoney bee ,61,62. Soney bee ,52,67. Money bee ,69,70,71oney bee  for reviPhacelia tanacetifolia to enhance pollinator visits close to apiaries of the domesticated Apis mellifera. To assess virus prevalence among bee species or functional groups, we detected common viruses originally described in honey bees  by multiplex PCR. In particular, under the assumption of a virus spillover among sympatric species foraging on the same resource, we would expect an increasing prevalence level in wild bees as time lapses since the onset of resource flowering. In addition, if viruses are transmitted between sympatric species, we would not expect host-specific clustering of the virus sequences from phylogenetic analyses.This study aims to (i) characterize the virus community shared by managed honey bees and wild bees foraging on the same floral resource, and (ii) identify potential evidence for virus spillovers between wild bees and a managed bee species. We used standard community ecology analyses to provide a general description of the shared virus communities among bee species and other flower-visiting insects. The experimental design was intended to enhance the putative spillover of viruses between pollinators sharing the same small surface of floral resource, and we planted the attractive but not endemic The overall experiment was based on the assumption that a virus spillover might be identified by monitoring, over time, the shared virus communities in bee species and functional groups that foraged locally on the same mass-flowering resource.Phacelia tanacetifolia, an attractive flowering crop often used in pollinator-friendly agro-environmental schemes [−1 sampling session per day, weather permitting (as soon as the temperature raised above 20 °C in the early morning), from the onset of flowering until its end. Along with each wild bee capture session, a sample of 40 foraging honey bees was captured and pooled for virus molecular analyses. Sampling began 5 to 8 days after the first flower was recorded (from Julian day 133 to 155) to allow for the first floral visits before sampling. Samples were collected at least twice a week throughout the blooming period . Individuals from the Halictidae family, belonging to Lasioglosum or Halictus genera, were pooled according to their size. Tubes were then stored at −80 °C. On each sampling day, captured wild bees were maintained dormant at 4 °C, identified to genus whenever possible, and ultimately assigned to homogeneous morphotypes intended to become monospecific individual pools. Identification was performed under a binocular microscope according to bee morphological keys for France and Western Europe. The identification keys of local interest are reviewed and provided for each bee family by the French expert naturalist association Osmia  to 15 individuals, which was the largest size our sampling regime could afford on a daily basis. Species pool homogeneity was subsequently confirmed from molecular evidence. 2, 43°54’57, 81” N, 4°52’33, 50” E) close to an apiary, thereafter referred to as the Yr1 pilot plot. Virus composition in pools of sampled wild bees was affected by pool size (see Results), underlying the need to standardize pool size. During the second experimental year (2015), as a trade-off between experimental resolution and productivity, we pooled individuals by 10 whenever possible in a daily session, and further favored single-individual samples otherwise. This strategy ensured a broad virus prevalence screening in a species-rich bee assemblage , and an in-depth virus prevalence focus on less common bee species .The first study year (2014) was dedicated primarily to set up a pilot study intended to validate the experimental design and to assess the most parsimonious individual pool sizes liable to reveal adequate virus prevalence in wild bees. As it appeared, on the one hand, unrealistic to obtain large monospecific pools for wild bees, and on the other hand, because we were unsure whether virus prevalence could be conveniently described using single individuals or few individuals per pool, we therefore favored an exploratory sampling strategy in a single pilot plot of phacelia (300 m2) 32 m away (Yr2 nearby plot), and a third 560 m2 plot, located 3.6 km away , bordering another apiary (Yr2 distant plot).We further included additional sampling plots to gain insights from spatial and temporal variations, aware of the variations that may occur over the season and across regions . Here, wg, and the supernatant was collected in a new tube to which chloroform was added (100 µL chloroform for 500 µL phenol/guanidine-based QIAzol Lysis Reagent (Qiagen), vortexed for 15 s and incubated at room temperature for 3 min. After 15 min centrifugation at 12,000× g at 4 °C, the aqueous phase was transferred for RNA purification on columns and treated according to the manufacturer’s protocol . The final suspension volume was 100 µL, and RNA was quantified on Nanodrop.A primary homogenate was prepared by grinding individuals pooled by morphotype Table S1A. mellifera samples, 40-individuals were pooled and RNA extracts were processed using standard methods, as described by Dalmon et al. [Xylocopa and Bombus individual wild bees were ground in 5 mL PBS 1× using plastic bags containing filter. Five hundred microliters of this primary homogenate (filtered extract) was added to 900 µL of Quiazol . After 5 min at room temperature, 180 µL of chloroform was added and samples were processed, as described above. For n et al. . Large s®, Carlsbad, Californie, CA, USA). Ten microliters from the resulting cDNA were used to perform a multiplex PCR reaction with 2.5 mM MgCl2, 10 pmol of each primer, [®  and 1 unit of Go Taq G2 Flexi DNA polymerase , in a 25 µL total volume, using an Eppendorf Mastercycler®  Nexus SX 1 or MJresearch PTC100®  thermocyclers. The following program was used: one cycle at 94 °C for 5 min, 35 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 45 s, and a final elongation cycle at 72 °C for 10 min. Amplified fragments code for part of the structural proteins, except for CBPV . Two negative controls (for reverse transcription and PCR) were included for each run. One microliter of samples from 2014 was analyzed using Agilent High Sensitivity DNA chips (Life Technologies®) in 2100 Bioanalyzer (Agilent®) . Because the expected specific bands were easy to separate , and did not require such a high sensitive electrophoregram, further PCR products (5 µL) were run in 4% agarose gel.Reverse transcription was performed from 1 µg RNA in a 20 µL total volume using random priming, according to the manufacturer’s protocol . 3 µL of the 10-fold diluted cDNA were added to 0.5 µM of forward and reverse primers, 2.5 mM MgCl2, 0.2 mM dNTPs, green buffer for GoTaq® and 1 unit of Go Taq G2 Flexi DNA polymerase , in a 25 µL total volume, and PCR conditions were the same as described above. Complementary sequencing was performed for DWV in the helicase coding region, using 5992f 5′-TCCTATTGCTGAATGTAGTC-3′ and 6693r 5′-GTTCACGACGCTTACTACAC-3′ [In total, 237 positive samples from the multiplex assay were amplified in a single PCR performed with specific primers of each virus to be sent to Genoscreen for purification and Sanger sequencing. Four samples from a French collection were added to phylogenetic studies (TACAC-3′  or in thTACAC-3′ . The folSequences were checked for homologies in data banks using BLAST. Maximum likelihood phylogenies were built using MEGA version 6 with boostrap resampling (1000 iterations). Sequences were submitted to Genbank under the accession numbers MW435683 to MW435746 (SBV); MW442594 to MW442650 (BQCV); MW442651 to MW442713 (ABPV); MW442714 to MW442727 (5′end of DWV); MW442728 to MW442746 (DWV-Helicase).2, 0.2 mM dNTPs, green buffer for GoTaq® and 1 unit of Go Taq G2 Flexi DNA polymerase (Promega), in a 10 µL total volume, and PCR was run with the following program: one cycle at 93 °C for 3 min, then 35 cycles at 95 °C for 30 s—51°C for 45 s—72 °C for 1 min and a final elongation at 72 °C for 8 min. Two microliters of the PCR products were run in 1.5% agarose gel to control the purity and band size, and then remaining PCR products were sent to Genoscreen for Sanger sequencing. Sequences were submitted to the BOLD database (http://www.boldsystems.org/index.php/IDS_OpenIdEngine) to obtain the species name. To identify the species of the insect captured, 4 µL of the 10-fold diluted cDNA (about 20 ng) were added to 0.4 µM of forward and reverse primers LCO-1490 and HCO-2198 , 1.3 mM All statistical analyses were performed with the R software for statistical computing . We first characterized the sampled bee communities. In a second step, we assessed the associated virus species assemblages using community ecology analytical approaches.vegan package [lme4 package, [Bombus species group was restricted to four common bumblebee species in our study, all displaying social habits with a queen and division of labour among workers. The halictid species group is usually composed of diverse, often small-sized, generalist bees with either solitary or social habits. All the other more rare bee species were pooled together for the sake of simplicity in the analyses of abundance variations. Honey bee abundances were not formally recorded because they were systematically present at saturating levels, as a result of the proximity of experimental apiaries, and always much more abundant than all other flower-visiting insects.We described the sampled bee communities in terms of species richness, composition and abundance across time and study plots. Species richness values were compared among plots using rarefied and extrapolated first-order Jackknife species richness estimator  and across functional groups using a negative binomial family distribution with a log-link function, which is recommended for count data that are subject to over-dispersion such as in capture surveys . Due to Variations of virus prevalence among bees sampled at different plots and times (days since the onset of flowering) were analyzed using an approach of multispecies distribution modeling , based on a multivariate GLM framework hereafter referred to as MGLM. Species distribution models are typically used to decipher how the distribution of a taxon (species) is driven by environmental variables. By handling multispecies datasets, MGLMs allow for the inclusion of many species simultaneously in a single multivariate model. It combines a species occurrence matrix (presence-absence in samples) and an environmental data matrix  and then uses permutation tests to identify potential species occurrence responses to the environment. Therefore, the species occurrence matrix is simply the virus presence table in bee samples, while the environmental data matrix is the descriptive table of those bee samples, i.e., the capture plot and day since the onset of flowering. Ultimately, virus occurrence predicted by MGLMs may be merely interpreted in terms of prevalence. Additionally, for wild bees, we included bee taxonomic assignations and functional traits such as bee sample characteristics liable to drive virus prevalence. Taxonomic variables were either the bee family, genus or species identity. Whenever a bee sample could not be assigned a species name based on COI molecular evidence, we kept the taxonomic resolution to the genus level. Functional trait variables were chosen based on their potential influence on virus prevalence, namely the level of sociality  social species) and the pollen diet specialisation .The potential associations between virus prevalence and bee sample characteristics were assessed in four steps, namely (i) a complete virus distribution model computation, (ii) a backward stepwise model simplification, (iii) a parsimonious model assessment and (iv) post-hoc univariate model confirmations. (i) Complete virus distribution model computation. We first fitted MGLMs for binomial presence-absence data with the traitglm function of the mvabund R package [ package . Complet(ii) Backward stepwise model simplification. Whenever a significant environmental effect was detected, we further simplified the model by dropping non-significant environmental variables. We used the Akaike information criterion (AIC) to guide model simplification, considering a trade-off between model fit and complexity. Model simplification was iteratively pursued until only a subset of significant environmental variables was included. The resulting model was therefore viewed as the most parsimonious candidate model.(iii) Parsimonious model assessment. As a final model simplification step, we recomputed the most parsimonious model using the LASSO penalty function that automatically drops irrelevant virus × environment combinations explaining the overall variations of virus prevalence models. This algorithm permits further highlighting of the most salient virus prevalence patterns throughout the many possible combinations.(iv) Post-hoc univariate model confirmations. We are first and foremost interested in the potential rise of virus prevalence as time lapses since the onset of flowering. Whenever MGLMs detected such a temporal pattern, we performed posteriori univariate confirmatory analyses, focusing on each virus species of interest separately. To do so, we used a GLMM framework for binomial family date, specifying the number of days since the onset of flowering as a fixed variable, while controlling for the non-independency of samples proceeding from the same sampling plot and year using the plot identity as a random grouping variable.A total of 2059 flower-visiting insects were captured and processed, among which 920 honey bees, 934 bees from genus Lasioglossum , 91 specimens of Bombus, 94 bees from 8 other genera or families, as well as 20 other non-apoid insects  and 265 for the other insects , unsuccessful COI amplification (n = 5) or species mixtures in individual pools as a result of incorrect morphotype assignment (n = 5 samples only). A total of 23 pooled samples of honey bees were analyzed  based on the first-order Jackknife richness estimator . Both thLasioglossum malachurum, a common social Halictid bee . The second most sampled species  was Bombus terrestris.The four local wild bee communities were largely dominated by n = 29 community samples out of 4 study plots, F = 1.67, p = 0.080), sampling dates since the onset of flowering , nor the interaction term . Species composition was found to be rather homogeneous across time and space, as the Permanova  returnedn = 90 daily samples out of 4 study plots, Chi2 = 11.8, p = 0.008). Interestingly, the post-hoc pairwise comparisons revealed that these variations were mostly explained by the lower bee abundances in year 2 in the pilot plot  as a random grouping variable. The overall wild bee abundance was significantly different among plots . The halictid bee abundance increase over time was consistent throughout the four plots , while IAPV and CBPV were less common —though with various prevalence levels. SBV and BQCV were highly prevalent  in the most common species , DWV was detected at low levels in Bombus sp, Halictus fulvipes and Lasioglossum malachurum , except syrphid flies . The backward stepwise model simplification remained inconclusive for temporal trends in the six viruses  as well as for plot effects .The virus distribution models computed with the multivariate GLM framework (MGLM) indicated that the overall virus prevalence in the honey bee samples was not dependent on the number of days since the onset of flowering, nor on the sampling plot  whose individuals nest alone, but close to other conspecific nests, and one cleptoparasitic species (Nomada distinguenda) whose individuals are solitary but lay eggs in the nest of other bee species. Those were represented by a single individual each. For the sake of simplicity, we assigned them to the solitary category. Considering pollen diet specialization, a single individual was formally identified as belonging to an oligolectic species , while all other species are known to be polylectic. This unfortunately precluded any meaningful analysis of virus prevalence based on diet breadth. Beside sampling days and plots, virus prevalence in wild bees could have been driven by social habits and dietary pollen specialization. We selected the single-individual samples for which COI species was confirmed and thus allowed us to obtain the complete functional information . This led to a subset of 103 samples belonging to 18 bee species from four families, with either solitary or social habits  were computed with all viruses, except CBPV, as it remained undetected in wild bee samples. We first performed the analysis on the 103 wild bee samples composed of a single individual. In a preliminary step, we computed complete models using either family, genus or species identity as candidate taxonomic drivers of virus prevalence. Family was by far the most parsimonious taxonomic resolution to account for virus prevalence, as it returned a much lower AIC value (ΔAIC >> 2) than the genus or species levels . n = 103 samples × 5 virus species, multivariate score = 126.3, df = 32, p = 0.004). However, the backward stepwise model simplification indicated that sociality was not a significant driver of virus occurrence. The most parsimonious virus distribution model included the plot effect, bee family, and days since flowering . Those L malachurum samples only displayed IAPV, SBV, ABPV and BQCV viruses. Here again, virus distribution models revealed that virus prevalence was highly dependent on the sampling plot  and days since flowering . The later temporal variations in virus prevalence were, however, not all significant, nor consistent with the temporal trends observed in single-individual bee samples , which notably included five more nucleotides in the intergenic region. Interestingly one Bombus terrestris sampled in 2012 in Northern France (12Bombus2-DeuxSevres 2012) was added to this study and clustered out of the Genbank sequences from other European countries, and out of the southern France sequence data from this study. Then, the French 2014–2015 strains from close locations are clearly distinct from the others in Genbank, which might suggest some geographic  structuring. A possible shared ancestry was investigated to look for a spatial and/or temporal structuring of the genetic diversity. Under the hypothesis of spillovers, virus isolates from different bee species are not expected to cluster separately as they share the same strains. All ABPV sequences from this study clustered in a large single clade , except n = 50 sequences) were too short (about 130 nucleotides) to produce a qualitative alignment and therefore no phylogeny could be built. However, similarities from BLAST clearly confirmed the assignation to IAPV species (data not shown). IAPV sequences (n = 63 sequences) were close to the European SBV sequences available in databanks, but clustered out of the Korea/Thailand group , and/or that this genomic region may be very conserved. One Bombus terrestris sampled in 2012 in Northern France (12Bombus2-DeuxSevres 2012) was added to the set of data, but clustered with reference accessions. From the 58 BQCV sequences (478 nt), no clear clustering could be observed  and Xylocopa iris (15X02) with A. mellifera and Eucera (14E09) sequences, but unfortunately the 5′ end of the genome could not be amplified for these wild species, neither from the few DWV positive Halictid pooled samples.DWV was highly prevalent in honey bees, and was also detected to a lesser extent in bumble bees, and in few other wild bee samples . The experimental design was set up to enhance and quantify processes of virus spillover among different groups of flower-visiting insects. Spillover, whereby a given virus is transmitted between two sympatric species or groups of species, is an example of indirect interference that can affect pollinators sharing the same foraging resources [While describing the viruses detected in both honey bees and wild bees sharing the same floral resource, possible spillovers between species were investigated by way of virus prevalence and virus sequence shared ancestry. From the 29 wild bee species sampled in this study (1137 specimens), we identified 22 species as potential hosts for viruses originally described in the honey bee, and five of them belonged to genera in which viruses had never been detected before, to our knowledge , 79% of the samples were positive for at least one virus, which is close to the prevalence already observed in other studies , as already observed for SBV in other studies  could have also contributed to an increase in the presence of viruses on the flowers. As a consequence, virus prevalence from this study has to be considered in light of the experimental design that intended to maximize the risk for flowers to be visited by an infected bee, while typical prevalence in other areas would be expected to be lower. That could contribute to the high proportion of co-infections we observed (about 51% of the samples with at least 2 viruses), while McMahon et al.  reportedl et al. ; 38% in l et al. ) when BQl et al.  when onll et al. ). In addl et al. . Levitt l et al.  did not l et al. . In addil et al. . Ants wel et al. . Then, vl et al. .8 virus particles per bee [Viruses may also be transmitted through physical contacts among individuals. Indeed, CBPV has the ability to be transmitted by contact. However, even if present in the honey bee, it was never detected in wild bees. CBPV occurred only in 17.4% of the honey bee samples, then this prevalence may be too low for allowing a contact between infected honey bees with wild bees. Furthermore, if multiplex PCR detected CBPV, no typical symptoms of trembling, paralysis and darkening of the honey bees were ever observed, and the viral loads may have been below the expression threshold of 10 per bee  and suchBombus spp. (less than 15% positive samples), and two social wasp Polistes spp., in accordance to previous findings in the UK [Vespula and Vespa genera [Lasioglossum malachurum, which was the most common wild bee species. Conversely, DWV may occur in carpenter bees Xylocopa spp. which are typically solitary—or with gregarious habits . But dii et al.  never foa et al.  suggesteL. malachurum , Vevelutina  and confbus spp. . All vir [Bombus ,105,106,velutina ,107, as velutina  and sevevelutina ,59. Finabus spp. ,104, andVespa velutina [Megachile rotundata and Colletes inaequalis by a mix lethal to honey bees  did not impact their survival, and virus titers decreased over time, suggesting that these viruses were not able to be replicated in these species [Additionally, the pathogenicity of the viruses on the potential new hosts remains to be investigated. From our sampling, we did not observe any symptom, while wing deformations associated with DWV have been observed in bumble bees  and Vespvelutina , and a nvelutina . Howevervelutina  could novelutina ,105, but species . HoweverA. mellifera could disseminate DWV. Other viruses  may be more ubiquitous than expected from their original description in the honey bees because host-specific clustering was never observed. With the increase of new virus descriptions using high-throughput sequencing, there is an urgent need to clarify the transmission dynamics of these putative pathogens, and to assess the risk they represent for pollinator communities. From the 29 wild bee species sampled, our study considerably extends the known host range of viruses originally described in the honey bee to five new genera and 22 species, detected positive for the first time, sometimes even from a few individuals. Sympatric species share viruses originally described in the honey bee, probably because flowers may act as transmission hubs. Host-switching may increase the risk to impact several wild pollinator communities. Directionality of the transfer is difficult to address from prevalence studies. However, based on both prevalence data and phylogenetic studies, we provide additional evidence that wild bees may act as reservoirs for some viruses, and that


Maize Chlorotic Mottle Virus (MCMV) in combination with any of the potyviruses namely; maize dwarf mosaic virus (MDMV), sugarcane mosaic virus (SCMV) and tritimovirus wheat streak mosaic virus (WSMV). In Africa, the disease occurs due to combined infections of maize by MCMV and SCMV, leading to severe yield losses. Efforts to address the disease spread have been ongoing. Serological techniques including enzyme-linked immuno-sorbent assay (ELISA), polymerase chain reaction (PCR), genome-wide association (GWAS) mapping and next generation sequencing have been effectively used to detect and characterize MLN causative pathogens. Various management strategies have been adapted to control MLN including use of resistant varieties, phytosanitary measures and better cultural practices. This review looks at the current knowledge on MLN causative viruses, genetic architecture and molecular basis underlying their synergistic interactions. Lastly, some research gaps towards MLN management will be identified. The information gathered may be useful for developing strategies towards future MLN management and maize improvement in Africa.Maize lethal necrosis (MLN) disease is new to Africa. First report was in Kenya in 2012, since then the disease has rapidly spread to most parts of eastern and central Africa region including Tanzania, Burundi, DRC Congo, Rwanda, Uganda, Ethiopia and similar symptoms were observed in South Sudan. Elsewhere, the disease was caused by infection of  Zea mays L., 2n=2x=20) is a major staple food and source of income and livelihood for the majority of smallholder farming communities in sub-Saharan Africa  and any of SCMV, MDMV  or WSMV  , DRC Congo , MCMV-KS (Kansas) and MCMV-YN (Yunnan), and different unconfirmed strains have been suspected in some parts of Africa including Nigeria, Rwanda, Sao Tome and Principe, Tanzania, Togo, Zambia and Zimbabwe  and aphid  are the common vectors of MLN viruses in Africa and the viruses can survive on different host plants such as cassava, beans, maize, sorghum, onions, rice, peppers coriander, peas, various grasses, Bidens pilosa and Tithonia diversifolia , southern corn rootworm (Diabrotica undecimpunctata), western corn rootworm (Diabrotica virgifera), Systena frontalis, Diabortica longcornis and Oulema melanopa , the only reported arthropod vector of MCMV, is the most common insect that transmit MCMV in most maize growing areas in Africa  and they function both as genome and messenger RNA. The viruses have evolved the ability to use the metabolic machinery of the host cell so as to produce their own genetic materials that they can use for multiplication and translation processes necessary for their survival as indicated in Viral particle enters host cell through wounds made mechanically or by vectors on the cell wall or by deposition into an ovule by an infected pollen grain  through replication by RdRp and translation of ssRNA(+) by ribosomes must occur on the same genomic template and at the same time. However, to avoid encounter between replication and translation apparatuses as well as collision of the two molecules as they move along the same template during the synthesis processes, ssRNA(+) viruses have developed a mechanism whereby ribosomes complete translation of ssRNA(+) before the negative strand replication by RP can start on the same template  in the surroundings are common signs and indications of potential MLN infections.Common symptoms of MLN include higher intensity of chlorotic spots, stunted growth and yellowing of the suspected maize plant . Phenotypic observation of symptoms of diseases alone cannot reveal the exact cause(s). Cells obtained from young leaf of virus-infected plants contain inclusion bodies of fairly distinctive shapes and sizes that can be seen using advanced tools so as to reveal the genus of the virus. Another important technique in detecting plant viruses is bioassay, which could be further confirmed by electron microscopy, serology and molecular techniques. Particles of many viruses are not always easy to find under the electron microscope; however, even when such particles are revealed, proof that the particles are of the virus that causes the particular disease requires much additional work and time  involved. Also, symptoms caused by the MLN viruses may vary according to the age of the maize plant, variety involved, environmental conditions, strain of the MLN viruses, and different viruses may cause similar symptoms on the same plant. Sometimes the disease symptom could result from co-infection by more than one virus , is easy to apply and is commonly used for detection of MLN viruses , and nucleic acid hybridization with radio-labelled and non-radio-labelled probes, and DNA/RNA probes  and polyclonal antibodies (PABs). The technique is widely applicable because ELISA based on RT-PCR method has some shortfalls since the method is more specific to a particular species or strain of a virus  and wheat streak mosaic virus  and they also infect maize, sorghum, wheat, oat, rye, johnsongrass and most species of Gramineae.Viruses of MLN are so complex and can survive and develop different strains or isolates across a wide range of hosts. Maize chlorotic mottle virus colonizes maize plant as the only natural host; however, the virus can experimentally infect grasses in the family Plant viruses are composed of minute building blocks (particles) called virions that make the complete viable structure. Shape of the virions differs from one virus to another and that is the main reason why viruses differ in their physical appearance when observed under electronic microscope as indicated in The genome is about 4-5.4 kb in length and about 30 nm in diameter. Both 5′ and 3′ terminals are protected by untranslated regions (UTRs)  as described in in trans. In addition, ORF2 produces P111 (111 kDa) protein by translational read-through of UAG stop codon found at its N-terminus. P111 is implicated in viral replication with some functions in trans. The function of ORF3 is not well understood  protein is expressed from ORF5 when the UAG stop codon of ORF4 is suppressed. The protein plays key roles in systemic infection. A 25 kDa coat protein (CP) expressed from the 3' proximal, is involved in cell-to-cell movement of virus. It is assumed that MCMV can overcome host defense mechanisms (RNA silencing) by expressing viral suppression of RNA silencing (VSRs) through P31, P32 and P50  encodes a 32 kDa protein which is assumed to be involved in viral accumulation. ORF2 (P50) encodes a 50 kDa protein and is associated with viral replication as well as function Genome of SCMV is 9610 nt long including the untranslated regions (UTRs) at both 5′ and 3′ terminals, which functions as infectious genome as well as messenger RNA, as presented in 4E at it C-terminal which interacts with eukaryotic translation initiation factors (eIF4E and its isoform eIFiso4E) by binding to siRNA or miRNA of the host, resulting to VSRs and enhanced viral infectivity  is expressed by autoproteolytic cleavage using serine protease and is involved in virus replication by stimulating helper component protease (HC-Pro) during VSRs. HC-Pro is synthesized through cleavage by cysteine protease. It is a multifunctional viral component which acts both as protein and enzyme (proteinase), and contains N-terminal, Central and HC-Pro domains. The N-terminal domain enhances virus transmission by aphids. It allows binding of virions to the cuticle of the maxillary food canal and foregut of aphids, virulence genome amplification and accumulation of virus  is a viral coat protein with major roles in virus movement, genome amplification and vector transmission. It is synthesized by the C-terminal of the polyprotein and is involved in encapsidation of viral genome using its multifunctional subunits  which is a small opening connecting adjacent cells , and polerovirus Beet western yellows virus (family Luteoviridae) has been reported with increased concentrations of all the three viruses and severe symptoms and stunting in sugar beet  plays major role in synergistic interactions as well as in replication and movement in SCMV. During co-infection of MCMV and SCMV, HC-Pro could be prioritized as VSRs, leading to reduction in SCMV replication and movement. Therefore, SCMV concentration in the mixed infections remains constant and being maintained by viral proteins such as P1 and VPg which are not strong enhancers of replication and movement proteins. This might explain why SCMV concentration in mixed infection with MCMV is unable to increase as for the MCMV  and sweet potato chlorotic stunt virus , the sweet potato plants showed severe symptoms in leaves and stunting of the plants. Concentration of SPFMV was increased 600-fold while that of the non-potyvirus partner (SPCSV) remained the same as in singly infected plants. This was because SPCSV was involved in VSRs of the host. As such, amplification and movement of SPFMV were enhanced  and both share some similar functions Scheets, . P1 may h-type thioredoxin responsible for resistance at Scmv1 locus, is reported to be involved in suppression of SCMV RNA accumulation  is generated and the Calvin cycle pathways within the photosystem I are affected. As a result, production of chlorophyll is reduced and MLN symptoms are expressed by the plant  gene, involved in COnt Cann, . Ferredont Cann, . Althougnt Cann, .A number of quantitative trait loci (QTLs) related to resistance to viruses have been reported in maize and so far genetic of resistance to MDMV, SCMV and WSMV is most studied of all viral diseases of maize  have been confirmed important and provide strong resistance against SCMV in maize. Scmv1 is responsible for protection against early infection while Scmv2 protects against late SCMV infections, and the two loci segregate as dominant genes , sugarcane, common weeds , and wild grasses. MCMV incidence is exacerbated in continuous maize production fields . However, it should be noted that the two scales 1-5 and 1-9 are similar because: 1=1; 1.5=2; 2=3; 2.5=4; 3=5; 3.5=6; 4=7; 4.5=8; and 5=9.A quantitative scale of 1-9 introduced by Reddy and Singh (1984) is also widely used based on diseased tissues relative to the whole plant: 1=resistant ; 2=resistant to moderately resistant ; 3=moderately resistant (mild chlorotic streaks on emerging leaves); 4=moderately to moderate resistant (moderate chlorotic streaks on emerging new leaves); 5=moderate resistant (chlorotic streaks and mottling throughout plants); 6=moderate to moderately susceptible ; 7=moderately susceptible ; 8=moderately susceptible to susceptible ; and 9=susceptible (complete plant necrosis and dead plants)  in collaboration with Kenya Agriculture and Livestock Research Organization (KALRO) has established an advanced MLN screening facility in Naivasha, Kenya of which partners including national research institutions (NARIs) have been screening their respective maize germplasm for resistnce to MLN viruses. The MLN screening facility uses artificial inoculation in order to create optimal screening conditions for MLN while ensuring minimal risk of disease escape. Optimized procedures have been developed to ensure uniform, high MLN disease pressure at the screening site (Olsen et al., Vast majority of African maize lines are susceptible to MLN and since the emergence of MLN in Africa in 2012, not much has been done on breeding for resistance to the deadly disease at national levels. CIMMYT, in collaboration with KALRO and other partners, has developed some maize hybrids and inbred lines that are tolerant to MLN. These materials are available for adoption by national research institutions (Olsen et al., Maize lethal necrosis disease is a new challenge to maize production and is threatening food security in Arica. For the last 4 years, most of the research interventions on MLN in Africa have been concentrating on etiology, epidemiology, screening and characterization of germplasm for sources of resistance to the disease (Adams et al., Each MLN virus exists in different strains and isolates; however, it is not known which combination of isolates gives severe MLN. Information on mode of inheritance of resistance to MCMV is inadequate. Multiple QTLs are linked to resistance to MLN; however, number of genes involved in resistance to MLN is not well reported. MLN is caused by co-infections of MCMV and SCMV; nevertheless, information on which virus to infect first so as to cause severe MLN synergism is lacking. Similarly, transmission of MLN through seed requires further investigation. Although a number of maize lines tolerant to MLN have been identified, their utilization in commercial hybrid is inadequate.MLN is a complex disease of maize with severe yield losses in Africa, hence its control requires effective and robust research approaches. A short-term strategy would be accessing tolerant lines from CIMMYT for adaptation in national programs. Mid-term research strategy may involve introgression of the tolerant genes into the susceptible, but farmers preferred maize varieties. In the long run, the breeding approach should corporate host resistance and vector management where transgenic breeding techniques could be employed.


The global burden of coronavirus disease 2019 (COVID-19) to public health and global economy has stressed the need for rapid and simple diagnostic methods. From this perspective, plasmonic-based biosensing can manage the threat of infectious diseases by providing timely virus monitoring. In recent years, many plasmonics’ platforms have embraced the challenge of offering on-site strategies to complement traditional diagnostic methods relying on the polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISA). This review compiled recent progress on the development of novel plasmonic sensing schemes for the effective control of virus-related diseases. A special focus was set on the utilization of plasmonic nanostructures in combination with other detection formats involving colorimetric, fluorescence, luminescence, or Raman scattering enhancement. The quantification of different viruses  with particular attention to Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) was reviewed from the perspective of the biomarker and the biological receptor immobilized on the sensor chip. Technological limitations including selectivity, stability, and monitoring in biological matrices were also reviewed for different plasmonic-sensing approaches. Viral infections can be transmitted among human populations in a short period of time via droplets, aerosols, contact with abiotic surfaces, and even fecal matter ,2,3. TheTraditionally, viral diagnosis has relied upon culture-based methods and serological tests that detect either direct (intact viruses or their components: proteins or nucleic acids) or indirect  virus infectious diseases .More recently, the advances of molecular techniques in viral diagnosis have permitted that nucleic acid-based amplification methods such as the polymerase chain reaction (PCR) and the reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) have consolidated as the gold standard methods for the routine diagnosis of a wide range of viruses (human immunodeficiency virus (HIV), hepatitis B and C viruses, and cytomegalovirus (CMV)) ,11,12,13Consequently, the requirement for fast and cost-effective viral diagnostic methods has led to putting the focus on the development of real-time biosensing platforms. Among the variety of biosensor technologies that have arisen in the last decades for virus detection, plasmonic applications have gained significant attention, owing to their versatility, label-free monitoring, and low time of response ,10,14. TOn the other hand, the chemical activation of the surface plays a major role for improving the sensing efficiency of single virus particles ,21. The Thus, nanoplasmonic biosensors provide a promising approach to attain ultra-low detection limits of viral particles, antigens, or nucleic acids from clinical specimens . The vast majority of virus sensing plasmonic applications are based on the well-known operation principles of surface plasmon resonance (SPR) biosensors. Nonetheless, the success in achieving optimal biosensing performance requires the design of novel biosensing strategies capable of maintaining the specificity and sensitivity of measurements while preserving the biocompatibility of the immobilized biological receptor.Therefore, the aim of this review was to present recent advances in plasmonic biosensing schemes for the detection of virus on the basis of signal enhancement. This review specially focused on the utilization of sensing surfaces comprising nanomaterials-based approaches. Current progress in the development of plasmonic-based applications involving the combination with colorimetric, electrochemical, fluorescence, or fiber-optic detection principles is also discussed.The fundamental principles of plasmonic biosensors rely on the propagation of surface plasmons along the interface of a thin, metal layer  and a dielectric (aqueous medium). A detailed description of the optical working principles was comprehensively reviewed in previous works and it is beyond the scope of this review ,23,24. IThis special characteristic allows enhancing the spatial resolution of LSPR configurations by designing the geometry and composition of metallic nanostructures, laying the basis for colorimetric plasmonic biosensing ,28. The Technological advancements in plasmonic biosensing including colorimetric and fluorescence enhancement as well as the utilization of nanomaterials and optical aperture nanostructures for achieving highly sensitive virus detection are described in this section.Nanomaterials provide the signal amplification of plasmonic biosensing due to their interesting optical, magnetic, and electrical properties. The biocompatibility and easy chemical activation are also significant advantages. Likewise, the large surface area-to-volume ratio provides a higher loading of biological receptors ,34,35,36Plasmonic nanomaterials can be classified regarding their chemical composition in organic  and inorganic  materials. Among the latter ones, noble-metal nanoparticles have been extensively applied to plasmonic biosensing due to the strong absorption of light resulting from the oscillation of the free electrons. The size and shape of nanoparticles along with the interparticle distance determine the amplitude of oscillation and the position of the SPR band . Likewis−1 [LSPR sensing is the most common sensing scheme that exploits noble-metal nanoparticles in analytical applications. LPSR biosensors employing gold nanoparticles (AuNPs) have been successfully applied to clinical diagnostics, environmental monitoring, and food safety . Particu−1 . First, Another LSPR platform takes advantage of a hollow Au spike-like nanoparticle (hAuSN) to detect avian influenza virus (AIV H5N1) using a multi-functional DNA three-way unction (3WJ) . This me−1, which is within the clinical levels observed in the first days of dengue infection. Despite the proposed method making use of spiked, whole blood samples instead of patient samples, the blood-plasma separation-integrated strategy represents a great advance toward the development of lab-on-chip devices and point-of-care virus diagnostics.The LSPR effect of silver nanostructures has also been reported to detect dengue NS1 antigen using an immunoassay format . The crePseudomonas aeruginosa and adenovirus [A singular LSPR immunosensor approach compares the capability of antibody-functionalized gold, silver, and copper nanoparticles for the detection of respiratory syncytial virus (RSV) in the presence of enovirus . The cha−1 levels.Plasmonic nanoparticles have also been exploited by SERS biosensors. The sensing scheme of SERS benefits from the enhancement of Raman scattering by providing the modulation of surface plasmon polaritons at a greater distance from the metallic nanoparticle surface, thereby offering higher sensitivity than that of SPR and LSPR. Lebedev et al. reported a SERS platform applied to the detection of viral DNA using nanoclusters (NC) of gold nanoparticles . SERS ex−1 with a limit of detection of 0.05 pg mL−1 . The applicability of the proposed method for clinical analysis was evaluated by testing HBsAg in spiked, real serum samples from nine patients showing recovery values between 96 and 104%.Another virus SERS-based strategy has been proven to detect hepatitis B surface antigen HBsAg . The bioHeavy metal quantum dots are semiconductor nanomaterials that have gained prominence in recent years for multiplex detection due to their broad adsorption and narrow emission band. The possibility of tuning the emission spectra over a wide range of wavelengths and the resistivity to external physico-chemical conditions are also significant advantages . Among t−1) and human serum (0.4 pg mL−1) (−1. The same quenching effect was observed when using CdSeTeS QDs/AuNPs’ nanocomposites for detecting norovirus-like particles (NoV-LPs) [−14 to 10−9 g mL−1 range, showing a limit of detection of 12.1 × 10−15 g mL−1. The selectivity for the detection of the target NoV-LPs was evaluated with influenza virus A (H3N2) and Zika viruses, demonstrating the nanobiosensor specificity  . The effNoV-LPs) . As the cificity .−1, depending on the composition of the nanohybrids: Alloyed AuAgNP-Qdot646-MB (molecular beacon) (1.7 copies mL−1) > CS Au/AgNP-Qdot646-MB (LOD = 2.4 copies mL−1) > AuNP-Qdot646-MB (LOD = 2.9 copies mL−1) > AgNP-Qdot646-MB (LOD = 7.6 copies mL−1)  . The worCarbon nanomaterials are graphite structures that can adopt different dimensions ranging from cylindrical nanotubes to plane nanosheets. Graphene-based nanostructures provide numerous benefits for plasmonic applications due to their physicochemical properties including a high surface and biocompatible area that facilitate surface functionalization ,61.Several SPR graphene-based applications have been developed by Mahdi’s group for the quantification of dengue virus at picomolar levels. A composite of reduced graphene oxide and polyamidoamine (PAMAM) dendrimers is described for the detection of the E-proteins of the serotype 2 of dengue virus (DENV 2). The detection format consisted of an immunoassay in which monoclonal antibodies were immobilized on self-assembled, dithiobis (succinimidyl undecanoate) amine-activated layers . The cha−1. Likewise, the same sensing scheme involving binary nanoparticle-graphene structures, although with the combined effect of gold and magnetic nanoparticles, was presented to detect norovirus-like particles [−1.Other plasmonic immunosensing approaches have exploited the potential of binary nanoparticle-graphene hybrid structures for the detection of virus in biological samples. For example, a magnetofluorometric platform made use of the combined effect of SERS and fluorescence of quantum dots to assist nanoparticle-decorated carbon graphenes in monitoring the presence of hemagglutinin (HA) protein in the surface of H1N1 influenza virus . The immarticles . In thisFinally, another composite consisting of gold nanorods and graphene oxide and gold nanorods (GO-GNRs) was applied to the determination of hepatitis B surface antigen using surface-enhanced Raman spectroscopy (SERS) as described above .The fabrication of plasmonic structures also relies on lithographic approaches, which enable the design of ordered arrays of metallic nanoapertures, such as nanoholes, nanoantennas, nanoslits, or nanodisks. The interaction of light with periodic arrays of nanoholes exhibits extraordinary optical transmission (EOT) effects, thus enhancing the transmission efficiency of light at certain wavelengths. These spectral features have facilitated the development of optical arrangements that support high bulk sensitivities and permit their integration into microfluidic systems and their application for the detection of single molecules ,63,64,65−1 levels, which significantly improved the recommended immunoassay-best test for the Ebola virus antigen detection.For instance, Zang et al. presented an interesting approach for single-molecule detection of Ebola virus antigens. The biosensor consisted of a 3D plasmonic nanoantenna array, which used a sandwich-immunoassay format with fluorescent intensity enhancement  Figure . The nanAnother plasmonic nanohole-based assay was developed for capturing single dengue virus-like particles . The dimSimilarly, another study measured the adsorption of a small virus particle of 25 nm (PhiX174) using double nanohole apertures in a gold film by the extraordinary acoustic Raman (EAR) method . As withSPR and LSPR biosensors that integrate optical fibers for coupling optical excitation of surface plasmons offer an interesting alternative to classical prism-based configurations. The possibility of driving the propagation of the electromagnetic field radiation through a fiber-optic system permits the improvement of the wavelength modulation and the development of compact structures. Many optical fiber SPR/LSPR platforms have provided miniaturized sensing approaches for the determination of clinical biomarkers . Most re4 to 108 EID 50/0.1 mL (EID: embryo-infectious dose), showing a detection limit of 5.14 × 105 EID 50/0.1 mL.In particular, a fiber-optic SPR system has been described for the analysis of avian influenza virus subtype H6 . To optiThe combination of the optical properties of gold nanostructures-LSPR surfaces with the achievement of total internal reflection via optical fiber configurations can provide signal enhancement and better spatial sensitivities. The integration of nanorod-modified surfaces into a fiber-optic LSPR platform have permitted the development of an immunosensor for the determination of a nonclinical application in plant viruses: Cymbidium mosaic virus (CymMV) and Odontoglossum ringspotvirus (ORSV) . The limFinally, the coating of an excessively tilted fiber grating surface (Ex-TFG) with gold nanospheres has permitted the development of an immunosensor for detecting Newcastle disease virus (NDV) . The modThe utilization of colorimetric assays is becoming more important in clinical diagnosis for detection of either organic  or inorganic molecules . The fabrication of plasmonic colorimetric nanosensors relies mainly on noble metal nanoparticles due to their high LSPR extinction coefficients in the visible range ,30. MostA plasmonic colorimetric sensor based on the aggregation of colloidal nanoparticles was described for the detection of influenza virus . The app−1 while H5N1 virus antigen was detected at 1 pg mL−1 levels using a sandwich-immunoassay format. The improvement in sensitivity in comparison with other methods was attributed to the high index faceted on the tips of the gold nanobipyramids with regard to gold nanorods.Another plasmonic colorimetric application for influenza virus detection (lineage H5N1) employed gold nanobipyramids (Au NBPs) as noble metal nanostructures . This ap2O2). A limit of detection of 10 pg mL−1 was obtained in buffer solution while the determination of HEV in real samples collected from fecal matter of HEV-infected monkey was in good correlation with RT-qPCR conventional methods .Similarly, a plasmonic platform consisting of silver deposition on antibody-conjugated gold nanoparticles was applied for monitoring hepatitis E virus (HEV) . A nanozFinally, a colorimetric assay approach based on the LSPR changes of silver nanoparticles induced by the hybridization chain reaction of the microRNA biomarker (miR-29a-3p) was described to detect H1N1 influenza A virus at picomolar levels . The hybThe effect of collective oscillations of free electrons on the surface of metal nanostructures displayed in LSPR sensing can enhance the optical signal of a luminophore near noble metal nanoparticles ,73. Plas3O4 yolk/Au shell (M@Au) was reported for the detection of a mouse sarcoma virus oncogene [3O4 nanoparticles and the stability and low toxicity of graphite quantum dots to detect nucleic acids in the 1-fM−1-nM range  as clinical nanosensors. For example, an ECL assay based on magnetic-plasmonic Feoncogene . The stunM range . The eff2 nanosheets to enhance the ECL signal of sulfur-doped boron nitrogen QDs (S-BN QDs) [2 nanosheets and S-BN QDs reduced the energy transfer while strengthening the surface plasma-coupling effect. Specifically, the assay was applied to the detection of hepatitis C virus (HCV) gene. The so-called hybridization chain reaction was used as an isothermal enzyme-free DNA amplification method to quantify DNA HCV with a limit of detection (LOD) of 0.17 pmol L−1.Another plasmon-based ECL strategy utilizes the strong surface plasmon-coupling nonmetallic MoS-BN QDs) . The appThe SARS-CoV-2 is an enveloped, nonsegmented β-coronavirus that causes a new, severe, acute respiratory syndrome and coronavirus disease COVID-19) [9 1,3,7575,78. SiNevertheless, the search for rapid and reliable point-of-care devices has triggered the development of plasmonic methods based on the latest technological advancements. For example, a novel approach combines the plasmonic photothermal (PPT) effect and LSPR sensing to detect DNA-selected sequences via hybridization to DNA receptors immobilized on two-dimensional gold nanoislands (AuNIs) . This du−1 of RNA with SARS-CoV-2 viral load. The main advantage of the proposed method was the possibility of being applied to target other regions of the viral genomic material, such as S-gene (surface glycoprotein), E-gene (envelope protein), and M-gene (membrane glycoprotein) without using sophisticated instrumental techniques.Another innovative approach for COVID-19 diagnosis comprised the development of a colorimetric assay based on gold nanoparticles (AuNPs) functionalized with thiol-modified antisense oligonucleotides (ASOs) specific for N-gene (nucleocapsid phosphoprotein) of SARS-CoV-2 . The bioThe uncertain proportions of pandemic outbreaks have triggered the need for reliable and cost-effective protocols easily adaptable to the changing virulence of virus strains. In recent years, plasmonic biosensors are being increasingly applied for clinical diagnosis of viral and other infectious diseases. Typical plasmonic biosensing strategies rely on the versatility of SPR and LSPR as label-free detection systems capable of monitoring binding interactions in a short period of time. Nevertheless, the incorporation of technological advancements has precipitated the development of nanomaterial-based applications for improving the sensitivity and specificity of classical configurations. The unique optical properties of plasmonic nanostructures has been exploited in combination with SERS colorimetric, fluorescence, or luminescence enhancement for viral diagnosis. Likewise, the development of plasmonic virus-sensing approaches has also benefitted from the variety of virus biomarkers. Thus, a high number of virus plasmonic biosensors have prompted the advance of novel functionalization strategies to achieve the effective coverage of the biological receptor while ensuring the affinity and specificity towards the target viral nucleic acids, proteins, or whole virus.Despite this substantial progress, current plasmonic applications have not yet outpaced conventional laboratory-based sequencing and immunoanalytical techniques. The improvement of sensing schemes has led to the attainment of ultrasensitive, robust, and reproducible analytical performances. However, the automation and integration of microfluidics as well as the prevention of nonspecific proteins’ adhesion in complex media still remains a challenge in many cases. On the one hand, the fabrication of automatable point-of-care devices is strongly dependent on both the design of reliable microfluidics and the integration of advanced software in miniaturized platforms. In this sense, the utilization of novel flexible materials via nanopatterning lithographic approaches may facilitate the incorporation of microchannels that enable the delivery of microvolume samples without requiring additional equipment. Secondly, plasmonic virus detection from clinical specimens is still limited due to the lack of universal methods that prevent the interference of biomolecules present in undiluted body fluids. From this perspective, the design of effective antifouling coatings, which take into account either the composition of the media or the biological receptor’s characteristics, will contribute to fill the gap between traditional analytical methods and plasmonics’ applications. By addressing these challenges, the huge potential for single virus detection along with the effectiveness and simplicity of current plasmonic configurations will impact on the routine surveillance of virus in clinical settings during this decade.


MCOLN1 gene, which encodes the late endosomal/lysosomal transient receptor potential channel TRPML1 (mucolipin 1). The precise pathophysiogical pathways that cause neurological disease in MLIV are poorly understood. Recently, the first post-mortem brain sample became available from a single MLIV patient, and in the current study we performed mass spectrometry (MS)-based proteomics on this tissue with a view to delineating pathological pathways, and to compare with previously-published data on MLIV, including studies using the Mcoln1−/− mouse.Mucolipidosis type IV (MLIV), an ultra-rare neurodevelopmental and neurodegenerative disorder, is caused by mutations in the Mcoln1−/− mouse by RNAseq, the results were remarkably similar. Finally, analysis of proteins in human and mouse CSF suggest that calbindin 1 and calbindin 2 might be useful as biomarkers to help chart the course of disease development.A number of pathways were altered in two brain regions from the MLIV patient, including those related to the lysosome, lipid metabolism, myelination, cellular trafficking and autophagy, mTOR and calmodulin, the complement system and interferon signaling. Of these, levels of some proteins not known previously to be associated with MLIV were altered, including APOD, PLIN4, ATG and proteins related to interferon signaling. Moreover, when proteins detected by proteomics in the human brain were compared with their orthologs detected in the Despite the sample size limitations, our findings are consistent with the relatively general changes in lysosomal function previously reported in MLIV, and shed light on new pathways of disease pathophysiology, which is required in order to understand the course of disease development and to determine the efficacy of therapies when they become available for this devastating disease. MCOLN1 gene, which encodes the late endosomal/lysosomal transient receptor potential channel TRPML1 (mucolipin 1) [MCOLN1 have been identified which lead to MLIV [Mucolipidosis type IV (MLIV), an ultra-rare lysosomal storage disease (LSD) primarily found in the Ashkenazi Jewish population , is causlipin 1) . Over tw to MLIV . TRPML1  to MLIV , 5. TRPM to MLIV , 7. MLIV to MLIV . At presMcoln1−/− mouse [Mcoln1−/− mouse is a genuine animal model of MLIV, and of more importance, allowed us to identify some novel pathological components and pathways in the human MLIV brain tissue, such as activation of complement and interferon (IFN) pathways and lipid droplet and lipoprotein metabolism. Along with the identification of a potential biomarker  for early disease development in the CSF, our study paves the way for further understanding of the molecular pathways that lead to disease pathophysiology in MLIV, a pre-requisite for development of putative therapies.We recently obtained brain tissue and cerebrospinal fluid (CSF) from a 36-year-old male who died from MLIV-related complications. This is the first time that post-mortem brain tissue has been made available from a MLIV patient. We performed mass spectrometry (MS)-based proteomics on this tissue with a view to delineating pathological pathways. Moreover, we compared our data with that obtained using a mouse model of MLIV, namely the /− mouse , 10, whiHuman brain and CSF samples were obtained from the NIH NeuroBioBank Brain and Tissue Repository at the University of Maryland, Baltimore. The post mortem MLIV patient was a 36 year-old male and the control was a 34-year-old healthy male who died from unrelated causes.www.uniprot.com) and appended with common laboratory protein contaminants. Quantitative comparisons were calculated using Perseus v1.6.0.7. Pathway analysis was performed using Gene Analytics [http://www.proteinatlas.org).Brain tissue was lysed using a GentleMACS dissociator  in 50 mM Tris HCl containing 5% sodium dodecyl sulfate (SDS) and supplemented with a protease inhibitor cocktail . Homogenates were incubated with 5 mM dithiothreitol for 1 h at 56 °C followed by 10 mM iodoacetamide in the dark at room temperature for 45 min. Samples were loaded onto S-Trap microcolumns  and subjected to tryptic digestion , 12. Samnalytics . Proteinnalytics  (http://www.uniprot.com), and appended with common laboratory protein contaminants. Quantitative comparisons were calculated using Perseus v1.6.0.7. Pathway analysis was done using Gene Analytics [Samples were lysed with 8 M urea and incubated at room temperature for 30 min prior to incubation with 5 mM dithiothreitol (Sigma-Aldrich) at room temperature for 1 h, followed by alkylation with 10 mM iodoacetamide (Sigma-Aldrich) in the dark at room temperature for 45 min. Samples were diluted in 2 M urea with 50 mM ammonium bicarbonate. Proteins were digested with trypsin   overnight at 37 °C followed by a second trypsin digestion for 4 h. Digestion was terminated using trifluoracetic acid . Peptides were desalted using an Oasis hydrophilic lipophilic balance column (Waters) and then vacuum dried and stored at -80 °C. The resulting peptides were analyzed using nanoflow liquid chromatography  coupled to high resolution, high mass accuracy mass spectrometry . Samples were analyzed in random order in discovery mode. The raw data was processed with MaxQuant v1.6.0.16  and analnalytics .Mcoln1−/− mice on a C57Bl/6 J background [Mcoln1/++ or Mcoln1±) were used as controls and both females and males were used in all studies. Genotyping was performed by PCR using genomic DNA extracted from mouse tails. Mice were maintained in the Experimental Animal Center of the Weizmann Institute of Science. Animal experiments were approved by the Weizmann Institute Institutional Animal Care and Use Committee.ckground  were useMcoln± or Mcoln1−/− mice were anesthetized using 200 mg/ml sodium pentobarbital and CSF aspirated from the cisterna magna using a glass needle. CSF samples were kept at -80 °C.1-, 2-, 3- and 7-month-old Mus musculus genome (mm10) using STAR. Only reads which mapped uniquely to genes were considered for further analysis. Differentially-expressed genes (DEGs) were selected using a 1.5-fold change cutoff between two populations and adjusted p value for multiple gene testing of < 0.05 [Brain tissue was homogenized using a GentleMACS dissociator and mRNA was isolated using the RNeasy mini kit . RNA concentration (ratio 260/230 and 260/280 nm) was measured using a NanoDrop ND-1000  and RNA integrity evaluated using an RNA screen tape on a Tapestation 2200 . A bulk variation of MARSseq  was usedf < 0.05 . Pathwayf < 0.05 . OrtholoCalbindin 2 was quantified using a Calertinin ELISA kit  according to manufacturer’s instructions. Samples were analyzed in triplicate.MS-based proteomics identified 3968 proteins in the cerebellum (CB) (with more than one unique peptide) and 3936 in the cerebral cortex (CRB). Of these, 959 were increased and 1512 decreased in MLIV compared to the control in the CB, and 824 proteins increased and 1168 decreased in the CRB (ratio > 1.5). 2113 proteins were detected in the CSF, with 1018 elevated (ratio > 1.5) and 526 proteins decreased in MLIV compared to the control. Due to the minimal sample size, label-free, relative quantification was calculated by dividing protein intensity in the MLIV sample by protein intensity in the control . When a Mcoln1−/− mice [To obtain an overview of changes in the MLIV brain, we first interrogated the proteomics data by pathway analysis. Identical pathways , as were some proteins associated with cholesterol metabolism , which were elevated in the CB but not CRB  with 174 up-regulated and 10 down-regulated. When genes detected in the mouse by RNAseq overlapped with orthologous proteins detected by proteomics in the human brain, the results were remarkably similar. Thus, levels of RNA encoding complement genes were elevated in 7-month old Mcoln1−/− mouse  were also detected at earlier pre-symptomatic stages, i.e. 1 month and early symptomatic (2/3-month old mice). Thus, even at early pre-symptomatic stages, levels of genes associated with the complement and IFN pathways were up-regulated. Likewise, microglia activation also begins early in disease development [Mpeg1, a macrophage marker expressed also in microglia, was DE in 1-month Mcoln1−/− mice, as was Cxcl10, whose levels are also altered in models of experimental autoimmune encephalomyelitis [In addition to proteomics analysis of human brain, we also performed RNAseq on the brain of −/− mice  at the ese Table , as wereelopment  as RNAsemyelitis .Mcoln1−/− mice, as were genes related to microglia activation [Trem2 and Tyrobp, key disease-associated microglia (DAM) genes [Levels of a number of other genes, including cathepsins and genes related to sphingolipid metabolism, were also up-regulated at early stages of disease in tivation  which inM) genes . Genes aM) genes  (Table 8M) genes , 27 and Mcoln1−/− mice. In the latter, 2092 proteins were identified and quantified. 67 proteins were DE (Mcoln1−/− versus Mcoln1±) at 1 month, 99 were DE at 2 months, 49 at 3 months and 162 at 7-months of age . While the same proteins were not detected at each of these ages, the deregulated proteins belonged to similar pathways. Thus, proteins related to a number of pathways increased, including lysosomal proteins , Ca+-binding proteins and proteins that regulate Ca+ levels , proteins related to the immune system , and several proteins associated with cellular trafficking . Levels of proteins related to apolipoprotein metabolism were decreased . As no common proteins were detected, we then compared all Mcoln1−/−mice to all Mcoln1± mice, independent of their age. 63 proteins were DE (48 up-regulated and 15 down-regulated). Levels of the Purkinje cell protein PCP4 were increased, as were levels of most lysosomal proteins , calbindin 2 (CALB2) and PCP4  and CLN3 patients [Since we were also able to obtain small amounts of CSF from the human MLIV patient, we performed a preliminary analysis to determine whether changes in the brain might be reflected in the CSF, and thus indicate that analysis of CSF might be useful to identify biomarkers. The CSF proteomics data was largely consistent with the brain data, but less so with CSF aspirated from patients . Due to patients . Thus, w. Our data confirm the usefulness of the Mcoln1−/− mouse as a genuine model of MLIV, and more importantly, shed light on some new putative players in MLIV pathology, such as apolipoproteins, lipid droplets, the IFN pathway, disease-associated microglia activation and autophagy. Finally, analysis of CSF from both the human patient and from Mcoln1−/− mice suggest that CALB1 and CALB2 might be useful as biomarkers to help chart the course of disease development and the outcomes of pre-clinical and clinical trials.We analyzed the proteome in two brain regions from an MLIV patient and a healthy aged- and gender-matched control, and annotated the observed changes in protein levels with changes previously observed in in vitro and in vivo models of MLIV