Journal of Virological Methods

Roseoloviruses, notably human herpesviruses 6A and 6B (HHV-6A and HHV-6B), are neurotropic viruses implicated as agents in neurological disorders, including: epilepsy, multiple sclerosis, and chronic fatigue syndrome. However, the effects of roseolovirus infection on neuronal signaling and network activity are not characterized. This is, in part, due to the complexities of monitoring electrical activity in individual neurons and across neuronal connections during viral infection. This protocol describes a human induced pluripotent stem cell (iPSC)-derived neuronal culture system that employs multi-electrode array (MEA) recordings to study neurophysiological changes during viral infection. Two culture platforms are described: (a) NGN2-induced forebrain neuronal cultures; and, (b) progenitor cell-derived neuron-astrocyte mixed cultures. Cell composition in cultures is validated by immunofluorescence staining using neuronal, glial, and viral markers. Functional activity is assessed using extracellular recordings detected via the MEA2100 system. Mean firing rate, inter-spike interval, single-electrode bursting, and network burst activity are characterized between roseolovirus-infected versus uninfected (control) states. Pharmacological treatment with bicuculline, gabazine, and nicotine in conjunction with immunofluorescence is used to confirm functional responsiveness of defined neuronal neurotransmitter chemotypes. Here, we show that HHV-6A infection changes neuronal firing compared to uninfected controls. The methods/workflows described provide a strategy to study how virus infection alters neuronal excitability and may be adapted to compare the effects of different viruses on nerve cell function, infection time courses, different multiplicities of infection (MOI), and therapeutic interventions.

Broad-range RT-qPCR assays are essential for detecting highly diverse viruses within virus families, but designing suitable primers remains challenging because of extensive sequence diversity and biases in public sequence databases. Here, we present a semi-automated workflow that combines sequence clustering and varVAMP to streamline pan-RT-qPCR primer design. The workflow reduces sequence redundancy, improves the representation of viral diversity, and facilitates the identification of broadly reactive primer candidates. We demonstrate the workflow using the arboviral genera Orthoflavivirus and Alphavirus as proof-of-concept datasets, showing its applicability to large and genetically diverse viral sequence collections. The workflow provides a practical, scalable, and reproducible framework for developing broad-range primer candidates and may support surveillance, diagnostics, and preparedness for emerging or divergent viruses.

Marmosets (MAR, Callithrix jacchus) are nonhuman primates extensively studied in a broad array of preclinical biomedical research areas. However, the limited number of available immunological reagents remains a critical shortcoming of these models. We successfully developed monoclonal antibodies (mAbs) pairs that can be used in immunoassays for the identification and quantification of the biomarkers of inflammation C-reactive protein (CRP), CXCL-10 (IP-10), interleukin (IL)-4, IL-6, and IL-10. Recombinant MAR protein variants were produced in mammalian cells, purified and used for mice immunization. Hybridoma clones were selected first for their capacity to bind the recombinant proteins. All possible pair combinations of these mAbs were then tested for their capacity to identify their targets in MAR-derived samples. These MAR samples consisted of supernatant of PBMCs exposed ex vivo to different stimulants, and plasma samples from animals stimulated in vivo with a low-dose intravenous LPS inoculation. The majority of the mAbs that recognized the recombinant proteins failed to bind to the MAR-derived homologs. However, we found MAR-derived reactive mAbs and selected pairs with optimal sensitivity and signal-to-noise ratio for immunoassays that included Luminex multiplexing assays, ELISA, and ELISpot assays. These immunoreagents and assays will improve the translational value of different MAR biomedical models.

Background. Vaccines have been an essential tool in bringing the world close to polio eradication, with over 99.9 percent of the global population free of poliovirus. No antiviral drugs or monoclonal antibody products, however, are currently licensed for treatment of polio. We previously isolated a human monoclonal antibody (9H2) with potent neutralizing activity against all three poliovirus serotypes. To advance 9H2 as a therapeutic candidate, we optimized its sequence to extend serum half-life and improve manufacturability. Methods. A Multi-Attribute Method under stress conditions identified post-translational modification sites, and Abacus (Trademark) ranked sequence liabilities. Six amino acid substitutions were introduced into the variable regions, generating 23 combinatorial variants. Codon-optimized genes were synthesized and engineered into a human immunoglobulin G1 backbone containing crystallizable fragment (Fc) mutations (M428L/N434S) to extend serum half-life. Constructs were transfected into CHO K1 cells to generate stable pools in 24 well plates. Protein A purified antibodies were characterized using biophysical assays and an in vitro poliovirus neutralization assay. Results. All variants retained high in vitro neutralizing activity against poliovirus. A lead candidate was selected based on integrated assessment of biophysical properties across eight assays and expression yield. Conclusions. Structure-guided engineering and experimental evaluation enabled optimization of the 9H2 antibody sequence and identification of a lead candidate suitable for clinical manufacturing as a potential anti-poliovirus immunotherapeutic agent.

Non-small-cell lung cancer (NSCLC) diagnosis is challenged owing to the need for reliable, non-invasive biomarkers. Circulating cell-free microRNAs (ccf-miRNAs) are promising candidates, but their low levels and short sequences in blood complicate detection. In this study, a novel nano-hybrid fluorescent array was developed to enable rapid and sensitive identification of NSCLC-related ccf-miRNAs. The array consists of poly(T)-modified fluorescent nano-polystyrene beads for capturing target miRNAs, and fluorophore-labeled, sequence-specific locked nucleic acid (LNA) probes for their detection. This design forms a sandwich structure with the target miRNA. Nano cytometry and fluorescence microscopy assessed both the capture and specificity of detection. The assay was tested on plasma samples for miR-16-5p and U6, evaluating selectivity, sensitivity, and reproducibility. The results showed that the poly(T)-modified beads efficiently captured the target miRNAs, and the LNA probes accurately distinguished the sequences. The assay enabled direct detection of miR-16-5p and U6 from plasma without amplification, demonstrating high selectivity, sensitivity, and reproducibility. Combining enrichment with nano-polystyrene beads and sequence-specific LNA probes addressed the main challenges of ccf-miRNA detection, namely their low abundance and short sequence length. As ccf-miRNAs are linked to NSCLC progression, this method could assist in early diagnosis, disease monitoring, and assessment of treatment response. The target-independent nature of the poly(T) capture layer allows for easy calibration to new miRNAs by changing the LNA probes, rendering it suitable for multiplex detection in clinical settings. However, further preclinical and clinical studies are needed before adoption in routine practice. This nano-hybrid fluorescent array presents a rapid and reliable approach for detecting low-abundance ccf-miRNAs in plasma. Its encouraging performance and flexible design show potential for future application in NSCLC diagnosis and monitoring, subject to additional confirmation.

BACKGROUND: The protein 4.1a/4.1b ratio is an intrinsic molecular marker of mean red blood cell (RBC) age resulting from progressive deamidation of protein 4.1b during RBC circulation. Despite its potential for estimating mean RBC age from a single blood sample, its application has been limited by labor-intensive RBC membrane preparation, and its interindividual variability has not been characterized in a large human population. METHODS: We developed a low-volume method for direct RBC membrane isolation from whole blood using saponin permeabilization and differential centrifugation without prior RBC washing. Membrane proteins were separated by SDS-PAGE, and protein 4.1a/4.1b ratios were determined by densitometric analysis with Gaussian peak fitting. Assay conditions, sample-volume requirements, membrane protein recovery, and analytical reproducibility were evaluated. The method was subsequently applied to 114 anonymized residual samples from routine diagnostic testing selected for complete blood count parameters within the corresponding laboratory reference intervals. RESULTS: The optimized procedure used 50 uL of whole blood, 0.06% saponin, and centrifugation at 9000 g, although protein 4.1a/4.1b remained measurable from starting volumes as low as 5 uL. Mean within-subject CVs were 3.90% for electrophoretic/densitometric determination and 5.39% for membrane protein recovery; whole-procedure CVs in 3 subjects were 1.40% to 3.50%. In the population analysis, the mean protein 4.1a/4.1b ratio was 1.428 (SD 0.146), with an observed range of 1.052 to1.799 and a central 95% interval of 1.151 to 1.695. Ratios did not differ significantly between females and males (P=0.246) or among females aged 18 to 50 years, females aged >50 years, and males (P=0.151). The ratio showed modest positive correlations with chronological age in females (Spearman p=0.26, P=0.047) and males (p=0.33, P=0.015), while substantial interindividual variability remained across the age range. CONCLUSIONS: Direct isolation of RBC membranes from small volumes of whole blood enables reproducible determination of the protein 4.1a/4.1b ratio using a simplified workflow. Application of the assay to a comparatively large human population revealed substantial interindividual variation in this molecular marker of mean RBC age that was not explained by sex or chronological age alone. The method provides a practical approach for investigating biological variation in mean RBC age and its potential contribution to laboratory measurements influenced by RBC survival.

Background: Hepatitis E virus (HEV) is highly endemic in several African countries with high mortality rate among pregnant women. The prevalence of HEV in Niger among pregnant women is unknown. We hereby aim to report the prevalence and risk factors of HEV among pregnant women in Niamey, Niger. Methods: Blood samples were collected from pregnant women attending antenatal consultation in three selected Health centers in Niamey. Data were collected for socio demographic characteristics using a structured questionnaire. Samples were tested using ELISA and qRT-PCR for anti- HEV IgM and RNA detection respectively. HEV associated risk factors were determined using multivariate logistic regression models. Results: A total of 491 pregnant women were enrolled with mean age of 26.5 years. The prevalence of anti-HEV IgM antibodies was 17.1%, while HEV RNA was detected in 3.46% of participants, indicating recent and active HEV infection. Anti-HEV IgM seropositivity was more frequent among women aged 26-30 years and those in the third trimester of pregnancy. Gravidity was significantly associated with HEV RNA positivity (p = 0.032). In multivariable analysis, the use of well water was independently associated with HEV infection (aOR = 75.70; 95% CI: 1.23-4643.20; p = 0.039). Conclusion: In view of the high seroprevalence of HEV among pregnant women, preventive measures including hygiene sensitization should be prioritized. Further studies should be conducted to assess the real impact of HEV infection on maternal and fetal health.

Orientia tsutsugamushi is the causative agent of scrub typhus infection in humans. This pathogen is transmitted into mammalian hosts including humans through Leptotrombidium mites. Till date, detection of O. tsutsugamushi in human was performed using PCR assay with 56kDa type specific antigen (TSA56), 47kDa outer membrane protein (47kDa-OMP) and groEL chaperonin gene. To understand the efficiency of different genes for the detection of O. tsutsugamushi in Leptotrombidium mites, we performed a comparative PCR assay with various universal primers targeting different genes including TSA56, 47kDa-OMP and groEL gene. We have collected 500 Leptotrombidium mites from Vellore, Tamil Nadu and, the mites were segregated into 50 pools with 10 mites in each pool were used for DNA extraction. The molecular detection of O. tsutsugamushi was performed using PCR assays (including conventional, nested and duplex PCR). The comparative PCR analysis showed nested PCR of groEL gene has high efficiency in detecting the presence of O. tsutsugamushi in chigger samples compared to other genes. Interestingly, groEL target demonstrated high efficiency for detection of O. tsutsugamushi in chigger samples and, when used with nested primers, can facilitate differentiation of O. tsutsugamushi from other rickettsial pathogens. Thus, we conclude groEL gene nested PCR assay can be used routinely to detect O. tsutsugamushi in trombiculid mites.

Human rhinovirus (HRV), a major cause of upper respiratory infections, has increasingly been associated with lower respiratory tract disease. Although tools to investigate HRV pathogenesis are needed, they remain limited. Reporter viruses expressing fluorescent or luminescent proteins are powerful tools for analyzing viral distribution and replication; however, insertion of a reporter gene often impairs viral replication, and the inserted gene is readily lost during serial passage. To address these problems, we established a versatile and stable strategy for generating reporter HRV and other enteroviruses. The gene encoding mScarlet3, a red fluorescent protein, was inserted between the 5'UTR and VP4 gene, a site previously used in other enteroviruses but not in HRV. Optimizing the 2A cleavage sequence between mScarlet3 and VP4 yielded a reporter HRV with growth kinetics comparable to those of the wild-type virus. To improve the genetic stability of the reporter insert, we evaluated GC-content optimization of the reporter gene and relocation of the cis-acting replication element (CRE). GC-content optimization substantially improved reporter stability in both HRV-B14 and EV-A71, whereas CRE relocation provided additional stabilization in EV-A71 but not in HRV-B14, demonstrating the potential applicability of this strategy to other enteroviruses. This strategy for constructing stable reporter HRV and other enteroviruses will facilitate studies of enterovirus pathogenesis.

Background: Amplicon-based next-generation sequencing is a cornerstone of viral genomic surveillance, but it often produces datasets with highly uneven read coverage due to PCR amplification bias. This leads to excessive data generation and substantial computational overhead for downstream bioinformatic analysis. Results: We evaluated the efficacy of two data reduction strategies on 220 SARS-CoV-2 clinical specimens and 12 wastewater samples: random subsampling with SeqKit and read depth normalization with BBNorm. FASTQ files were reduced to target depths from 10X to 10,000X to compare genome coverage, lineage assignment concordance, and computational resource usage. Both strategies markedly reduced total processing time. For clinical isolates, read depth normalization corrected for amplification bias resulting in highly concordant lineage assignments at a target depth of just 200X. Random subsampling preserved inherent bias but produced reliable assignments at depths over 500X. Reducing target depths below 500X in complex wastewater mixtures severely degraded variant deconvolution accuracy and heavily skewed lineage abundance profiles for both methods. Conclusion: Both random subsampling and read depth normalization successfully reduce computational overhead while preserving key clinical sequencing metrics at appropriate reduction settings. The optimal method depends on the analytical goal, ranging from computationally lightweight random subsampling for rapid lineage assignment, to read depth normalization for generating high-quality consensus sequences. However, aggressive data reduction should be avoided in environmental surveillance, where maintaining a high baseline sequencing depth is critical for accurate mixed-variant deconvolution.

Recent zoonotic coronavirus outbreaks have sparked an interest in understanding coronaviruses circulating in animal reservoirs. While bats are recognised as major reservoirs for these viruses, coronaviruses have also been detected in a wide range of terrestrial small mammals, particularly rodents. However, coronavirus diversity in these mammals, specifically in Europe, is still poorly characterised. In this study, we detected and characterised coronaviruses in terrestrial small mammals and bats from Belgium, France, Germany, and Ireland. We screened tissue samples from rodents and shrews and environmental bat guano using pan-coronavirus real-time and conventional PCRs, with positive results confirmed by sanger sequencing. Among 2667 bat and 1152 terrestrial mammal samples, we detected 59 positive samples, 36 from bats and 23 from rodents, from which we recovered 60 coronavirus sequences, spanning both Alphacoronavirus and Betacoronavirus lineages. Through next generation sequencing we identified one Pipistrellus pygmaeus bat guano sample carrying two distinct alphacoronaviruses, representing Nyctacovirus and Pedacovirus subgenera. Among rodents, most coronavirus-positive samples originated from a zoo in Belgium, where several CoV lineages co-circulated. The identified rodent coronaviruses clustered among previously described rodent-associated lineages, some of which also include human and other mammal-derived coronavirus sequences. Consistent with previous studies, we found sarbecoviruses in horseshoe bats (particularly Rhinolophus ferrumequinum and R. hipposideros) and merbecoviruses in Plecotus auritus bats, and these viruses are distantly related to the human coronaviruses of the respective families. Similar to bat coronaviruses, some rodent coronaviruses strains seemed to be association with specific rodent species. In conclusion, our study enhances the understanding of coronaviruses in rodents and bats, revealing potential hotspots for these viruses and their implication for human and animal health. These findings underscore the necessity for continued surveillance of coronaviruses in wildlife reservoirs to mitigate future zoonotic spillover risks.

Plant-derived flavonoids show measurable anti-dengue activity in cell culture, but their translational value is limited by a narrow therapeutic window: the concentrations that inhibit the virus approach or exceed those that are cytotoxic. Whether nanoparticle formulation can resolve this constraint, rather than simply add potency, remains untested for a chemically defined fraction. Here we show that biogenic silver nanoparticle (AgNP) formation using a flavonoid-enriched fraction of Carica papaya inverts an unusable selectivity profile into a viable one. The unformulated fraction was cytotoxic below the concentrations required for antiviral activity: its 50% cytotoxic concentration (CC50 = 134.9 ug/mL) lay below its 50% effective concentration against dengue virus serotype 2 (DENV-2; EC50 = 254.4 ug/mL), giving a Selectivity Index (SI) of 0.53. Using the same flavonoids as sole reducing and capping agents produced AgNPs (Z-average 128.4 nm; PDI 0.232; zeta potential -28.4 mV) that moved both parameters simultaneously. CC50 rose approximately 8.6-fold to 1,165.74 ug/mL while EC50 fell approximately 8.8-fold to 29.05 ug/mL, raising the SI to 40.12, an approximately 75-fold shift. Time-of-addition analysis localised the effect to the extracellular phase: inhibition was significant under pre-treatment and co-treatment but not after viral adsorption, identifying the AgNPs as entry inhibitors rather than replication inhibitors. Consistent with a serotype-independent physical mechanism, AgNP treatment at 30 ug/mL reduced viral RNA across all four serotypes, using inocula standardised against WHO-traceable NAAT reference reagents. These findings identify capping chemistry, rather than silver content alone, as a determinant of the therapeutic window in phytosynthesised nanoantivirals.

Neurologic autoantibody cell-based assay images are difficult to interpret when fluorescence is weak, focal or dominated by background signal. We developed a visual artificial intelligence overlay that transfers detected cell boundaries from the structural channel to the antibody-staining image and evaluated standalone model concordance and its association with reader classifications. The analysis used 2 763 records for model development and 499 held-out records spanning 14 antibody targets. Four readers assessed the held-out records first without assistance and then with the boundary overlay and model category available. The selected model agreed with the reader-derived three-class reference in 85.2% of records, but recall was lowest for weak-positive or equivocal records. Reader agreement increased in the assisted session, and almost all revised ratings moved towards the model category. The combined display may support consistent visual review while also creating a risk of model-directed anchoring; its role as a second-read aid requires independent validation.

Low pathogenic avian influenza A(H7N7) virus detected in a poultry worker in Taiwan, 2026 was related to viruses circulating in Asian wild birds and possessed PB2 E627K, a mammalian adaptation marker. Enhanced surveillance for H7 viruses in poultry and rapid characterization of human cases are essential to assess pandemic potential.

Macrolides remain the first-line antimicrobial treatment for pertussis, primarily caused by Bordetella pertussis. Emergence of macrolide-resistant B. pertussis in Australia and globally presents a growing concern for clinical management. As molecular diagnosis predominates for pertussis, limited numbers of cultures are available for antimicrobial susceptibility testing. The A2037G mutation in the 23S rRNA gene is most commonly associated with macrolide resistance in B. pertussis, yet no commercial molecular assay is available for its detection. In this study, we adapted and validated a previously published real-time PCR assay as a laboratory developed test (LDT) on the Panther Fusion(R) system using Open Access functionality to detect the macrolide resistance-associated mutation in B. pertussis from culture isolates and clinical specimens. Total turnaround time was ~2.5 h for the first result and 2-3 min for subsequent results in the same batch, with an estimated cost of AUD 18 per sample, excluding labour. A total of 30 samples were included in the validation panel to assess assay performance. All isolates and clinical specimens in the validation panel produced results concordant with the expected mutation status, demonstrating 100% accuracy, sensitivity and specificity. The assay demonstrated a limit of detection of 1.5 x 103 CFU/mL (22 CFU/reaction). This Panther Fusion(R) LDT provides a rapid and fully automated approach for molecular detection of the targeted mutation associated with macrolide resistance in B. pertussis. Its integration into routine molecular workflows as a reflex test following identification of B. pertussis may facilitate timely identification of resistant infections.

In the enzymatic toolbox of depollution, hydrolytic dehalogenases are of particular interest for the bioremediation of organohalogenated compounds. To better identify and characterize these enzymes, it is important to develop appropriate sensing methods. Fluorescent probes for dehalogenase detection have been developed in the past years, however, to this date none were available for application using the common FITC parameters (i.e. excitation laser at 488 nm). To address this limitation, we herein report a new fluorescent probe, COU4-Cl, excitable in the violet (405 nm) and blue (488 nm) wavelengths adapted for the detection and characterization of haloalkane dehalogenases. Its applicability is discussed and evaluated in comparison to the previously described COU2-Cl probe only excitable in the violet (405 nm). This study shows the advantages and complementarity of the two probes, and further demonstrate their application for the discrimination of dehalogenase activity in whole cell assays.

Numerous CRISPR-Cas systems have been developed for molecular detection of genetic elements exploiting the trans-cleavage activity of LbCas12a combined with short fluorescent probes. Alongside, a large variability of buffer conditions has been reported. However, how solution chemistry balances enzymatic turnover, signal stability, and detection performance have not been systematically defined. Here, we evaluated how the interplay between anions (chloride vs. acetate), additives, sequence probes, and ionic strength contributes to LbCas12a trans-cleavage activity. Buffer composition screening revealed that chloride-based buffers maintain a low background but slow catalytic rates, meanwhile acetate-based buffers accelerate enzymatic turnover with and without the target DNA sequence, increasing non-specific signals. Reducing agents and surfactants were the best enhancer combinations, improving cleavage performance by increasing ternary complex formation (Km). High salt concentrations only slightly improved the reaction kinetics. Finally, the 5FAM-TTATT-3BHQ-1 reporter showed the best balance between signal intensity and nonspecific background. Our findings indicate that LbCas12a tolerates a vast buffer composition landscape, maintaining robust trans-cleavage activity and detection performance. However, conditions that enhance catalytic activity simultaneously increase nonspecific probe cleavage and higher background, resulting in lower detection performance. Therefore, assay optimization should prioritize detection performance rather than maximal fluorescence output, where solution chemistry is a critical determinant.

Powassan virus (POWV) is an emerging tick-borne orthoflavivirus that causes severe encephalitis, long-term neurologic sequelae, and death, yet no approved antiviral therapies exist. Here, we evaluated several previously characterized nucleoside analogs as candidate countermeasures for POWV encephalitis. Favipiravir, molnupiravir, 4'-fluorouridine (4'-FlU), and GS-441524 inhibited POWV replication in vitro and demonstrated comparable activity against West Nile virus (WNV). We established C3H/HeNCrl mice as a stringent POWV disease model, showing increased susceptibility, higher day 7 brain titers, and more pronounced neurologic signs resembling severe human disease, compared with C57BL/6 mice. In prophylactic and therapeutic studies, 4'-fluorouridine was the most efficacious compound tested, significantly reducing brain viral burden even when treatment was delayed. Direct comparison with molnupiravir, favipiravir, and GS-441524 in mice confirmed superior survival benefit and antiviral activity for 4'-FlU against both POWV and WNV. These data identify 4'-fluorouridine as a promising therapeutic candidate for neurotropic flavivirus disease.

Background: Klebsiella pneumoniae is a key causative agent of multidrug resistant nosocomial infections. Bacteriophages offer a promising alternative treatment, but the diversity of Klebsiella species requires the characterisation of a broad range of phages, particularly the poorly characterised jumbo phages. Materials and Methods: Phage BMCPR_005026 was isolated from sewage against Klebsiella pneumoniae. Whole genome sequencing, transmission electron microscopy and host range analysis against a diverse set of 40 clinically relevant Klebsiella strains were used to characterise the phage. Results: The virion had a myovirus morphology and genomic analysis placed it within an undefined family in Caudoviricetes where it forms a new species within the genus Alcyoneusvirus. BMCPR_005026 could form plaques on 3 of the 40 Klebsiella strains tested (7.5%) and formed halos. Conclusion: BMCPR_005026 represents a new species of phage within the genus Alcyoneusvirus with activity against 3 Klebsiella KL-types.

The apoplast is a primary location for interactions between plants and invasive pathogens and is the site where many pathogen-secreted effectors are perceived by cell surface immune receptors to activate defence responses. However, our understanding of apoplastic interactions remains limited because protein interactions are difficult to investigate in this harsh extracellular environment. TurboID-based proximity labelling (PL) has emerged as a powerful approach for studying protein interactions in plants, though its application has to date been restricted to intracellular proteins. Here, we designed and validated a TurboID-based PL strategy for investigating protein interactions in the leaf apoplast using the well-characterised interaction between the Phytophthora infestans elicitor INF1 and the receptor-like protein (RLP) REL in Nicotiana benthamiana. Transient expression of SP-INF1-TurboID (INF1-T) induced a cell death (CD) response comparable to that triggered by native INF1, demonstrating that fusion of the TurboID tag did not impair INF1 recognition by REL. Apoplastic localisation of both INF1-T and the control construct SP-eGFP-TurboID (eGFP-T) was confirmed, validating their suitability for PL experiments. Efficient TurboID-mediated biotinylation was achieved in the apoplast using co-infiltration of biotin, ATP and magnesium acetate. Streptavidin-HRP immunoblotting revealed distinct biotinylation profiles for INF1-T and eGFP-T. Furthermore, co-immunoprecipitation demonstrated specific biotinylation of REL by INF1-T, but not by eGFP-T, in wild-type, bak1 and sobir1/sobir1-like N. benthamiana. These findings demonstrate that TurboID-based PL is functional in the apoplast and provides a proof-of-concept for investigating elicitor-receptor interactions in this compartment.