Monkeypox was re-emerging in 2022 and spread to more than 100 countries. Two clades of Monkeypox virus (MPXV) result in different lethality rates and varying transmission capabilities. Rapid identification of MPXV and differentiation of its clades and subclades are crucial for effective control of the disease. In this study, we developed an ARMS-Quadruplex-qPCR method to detect MPXV and distinguish clades (Ia, Ib, IIa and IIb). F3L gene was used to detect all clades of MPXV from other orthopoxviruses. A 1953 bp fragment containing the C3L gene was found to be completely absent in clade II. Additionally, a sequence spanning from the 177th to the 1318th position (1142 bp) within the 1953 bp fragment was missing in Ib. Therefore, the 1142 bp sequence was used to distinguish Ia from other subclades, and the sequence with the 1142 bp region missing in Ib was used to discriminate Ib from other subclades. Since subclades IIa and IIb are too close to have large deletions and insertions, a unique single nucleotide polymorphism (SNP) was used to design a primer/probe set for ARMS-qPCR to differentiate clade IIa from IIb. The ARMS-Quadruplex-qPCR system can detect down to 2 copies per reaction of MPXV and effectively differentiate all the four subclades. Altogether, four qPCR primer/probe sets in one tube were deployed to recognize MPXV and differentiate MPXV subclades. The high sensitivity, rapidity and specificity of the developed system make it a promising alternative for the diagnosis of MPXV and the determination of the subclades of the infected MPXV.

Human papillomavirus (HPV) infection is a major global health challenge and is closely related to the occurrence of diseases such as cervical cancer. Unfortunately, effective treatments are still lacking. In view of the advantages of antibody drugs, antibody-targeted therapy may become one of the means of treatment and prevention of HPV infection. This study explores the potential of antibody-targeted therapy using immunization with HPV nine-valent vaccine in Leghorn chickens. The resulting egg yolk antibodies (IgY) was extracted from eggs using the bitter-ammonium sulfate method and confirmed through SDS-PAGE analysis. The neutralizing titer was performed by pseudovirus-neutralizing antibody experiments, which could reach 1:2000 (18.2 μg/mL). This successful preparation of IgY against HPV 6/11/16/18/31/33/45/52/58-L1 protein showed its potential as a therapeutic agent, particularly post-HPV16 infection. This work lays the groundwork for HPV-specific IgY preparation and contributes to advancing targeted therapies for cervical cancer, prompting further research in HPV-related therapeutic approaches.

During the COVID-19 pandemic, reagents for SARS-CoV-2 detection were scarce or sold at high prices, particularly in Latin America. In this study, a significant step towards self-sufficiency was achieved through the development of an in-house extraction kit for detecting SARS-CoV-2 from nasopharyngeal swab samples. The purity and concentration of the RNA extracted using the in-house kit were compared to those obtained using the GeneJET RNA Purification Kit (Thermo-Scientific®) as a reference. The applicability of the RNA extracted using the kit was evaluated using four samples positive for SARS-CoV-2 by NGS sequencing with Illumina®. There were no significant differences between the results obtained with the in-house kit and those obtained with the commercial kit. These findings confirm that the in-house protocol demonstrated satisfactory diagnostic accuracy for detecting the virus in patients with COVID-19. The in-house extraction kit works effectively, providing optimal RNA extraction for genomic characterization and lineage assignment of SARS-CoV-2 within the four positive samples analyzed. This phenol-free kit represents a local design and production achievement, offering an effective solution for RNA extraction and detection and sequencing of SARS-CoV-2 from nasopharyngeal swabs. The data highlight the essential contribution of this study to health and biotechnological sovereignty in Colombia.

Unprecedented H5N1 highly pathogenic avian influenza (HPAI) outbreaks are occurring around the world and there is growing interest in the use of vaccines in affected regions. Vaccination when properly applied can contribute to HPAI control by significantly reducing virus shedding and breaking the transmission chain, but it requires robust surveillance to ensure that international trade is not affected. Thus, it is imperative to establish a test to differentiate vaccinated only animals from vaccinated and then infected animals (DIVA). In this study, we applied enzyme-linked lectin assay (ELLA) to specifically detect N1 neuraminidase (NA) antibody by inhibition of NA activity and provide a proof-of-concept bench validation using reference and experimental serum samples. We used a wild-type low pathogenic H7N1 virus of North American lineage as the ELLA antigen. The NA inhibition ELLA (NI-ELLA) was evaluated for its specificity and sensitivity using reference and experimental samples. The results demonstrated that the NI-ELLA was highly specific with low background NI activity against influenza-negative sera from different species although varying level of cross-reactivity was observed against sera of different NA subtypes with highest cross-reactivity against N4 subtype sera. Using a conservative positive cut-off threshold of 50 % NI activity, NI-ELLA provides 100 % specificity with all reference sera of 9 different NA subtypes. The relative sensitivity of NI-ELLA was evaluated in detecting H5N1 infection in vaccinated and then challenged birds and NI-ELLA showed higher detection rate of H5N1 infection compared with commercial NP ELISAs and real-time RT-PCR. Overall, the NI-ELLA shows high specificity and sensitivity and has the potential for application in DIVA surveillance with further validation.

Viral hemorrhagic septicemia virus (VHSV) is a significant pathogen causing mass mortalities in marine and freshwater fish worldwide. Accurate diagnosis through quantitative reverse transcription PCR (RT-qPCR) is essential to prevent its spread, but false negatives can compromise results. In this study, we evaluate the use of heat-inactivated snakehead rhabdovirus (SHRV) as an internal positive control (IPC) for VHSV diagnosis. SHRV's similarity to VHSV in viral structure and genome makes it an ideal IPC. The introduction of SHRV IPC into the RT-qPCR workflow can improve the reliability of diagnostic results by enabling the detection of technical failures during the RNA extraction or amplification process. Our results show that heat-inactivated SHRV preserved RNA integrity for IPC use, and SHRV IPC can provide a useful tool for detecting and interpreting false negatives without affecting assay sensitivity.

Sacbrood virus (SBV) is a significant pathogen affecting honeybee health, leading to substantial economic losses in apiculture. Although traditional methods, like reverse transcription quantitative polymerase chain reaction, offer accurate detection and quantification of SBV, they have limitations for use in field settings, such as apiaries. Here, we developed and evaluated the XQ SBV Dx Kit as a diagnostic tool for the XQ Station point-of-care (POC) RT-qPCR device, which integrates nucleic acid extraction, gene amplification, and detection for on-site molecular diagnosis. Diagnostic performance was assessed using clinical samples infected with SBV and was compared with that of standard laboratory-based RT-qPCR. The limit of detection (LOD) for both methods was 10 copies per reaction, with the XQ SBV Dx Kit consistently demonstrating superior sensitivity, detecting 83.3 % of replicates at 10 copies per reaction compared to 58.3 % with RT-qPCR. Specificity testing against 11 other honeybee pathogens confirmed the absence of cross-reactivity, highlighting the diagnostic precision of the XQ SBV Dx Kit. Clinical evaluation revealed 98.4 % sensitivity and 97.0 % specificity, validating its reliability for field applications. Overall, the XQ SBV Dx Kit is an essential advancement in honeybee health management, offering practical and timely solutions for supporting sustainable apicultural practices.

HIV-2 affects over 1 million people globally and can lead to AIDS if untreated. Treating people living with HIV-2 (PLHIV-2) is challenging because the virus is inherently resistant to some drugs. Effective treatment monitoring, particularly drug resistance testing, is critical for managing therapeutic failure. Without commercial tests to identify drug resistance mutations (DRM), laboratories have felt the need to develop in-house methods. NGS provides improved sensitivity for detecting minority DRM, which is crucial for effectively treating individuals, especially with limited therapeutic options. This study aimed to evaluate the effectiveness of a hybrid NGS Ion Torrent protocol for the detection of DRM in PLHIV-2 and its use in clinical practice. One hundred samples from PLHIV-2 collected from hospitals across Portugal were analyzed using a hybrid NGS protocol. Of these, 48 samples were also subjected to Sanger sequencing for comparative purposes. NGS successfully amplified 92 % of protease, 91 % of reverse transcriptase, and 49 % of integrase regions. The two sequencing methods agreed on the majority of DRM identified, with the only difference in two samples for the reverse transcriptase, which NGS identified as K70E and M184V, while Sanger did not. Hybrid NGS was able to identify DRM, demonstrating strong statistical agreement. In conclusion, hybrid NGS detected all DRM identified by Sanger, with the added ability to detect minority variants. The implementation of NGS-based protocol can provide clinicians with more comprehensive data, allowing for adjustments to ART regimens, and ultimately improving patient outcomes and quality of care for PLHIV-2.

The Zika virus became a global threat in 2015 due to its association with microcephaly. Preventing its spread depends on developing vaccines, with virus-like particles (VLP) being a promising approach, especially because of their safety profile and high immunogenicity. This study focused on the production of Zika VLP using Sf9 cells and the baculovirus expression system, evaluating cell growth kinetics, nutrient consumption, and metabolite production in Sf-900™ III medium. As a methodology, this study includes bioreactor experiments, cell density and viability quantification, nutrient and metabolite analysis, Dot Blot, Western Blot, and transmission electron microscopy. Among the critical conditions tested are culture medium supplementation with 0.028 mM cholesterol/ 6 nM bovine serum albumin, multiplicity of infection (MOI= 0.2 or 2), and dissolved oxygen tension (DOT= 5 or 30 % air saturation). As a result, in the growth phase, Sf9 cells achieved rapid exponential growth, with doubling times ranging from 22.8 to 35.4 hours and standard nutrient consumption and metabolite generation profiles for this cell line. The infection phase recorded cell death rates between 8200 and 12600 cells mL⁻¹ h⁻¹ , with higher VLP production under low MOI (0.2) and low DOT (5 %). These conditions also reduced protein degradation and nutrient consumption. The produced VLP ranged from 32 to 73 nm in size, with smaller sizes observed under low MOI conditions. Finally, controlling the DOT at 5 % air saturation without cholesterol/albumin supplementation increased VLP production without the need to raise the viral load, highlighting the importance of choosing the appropriate combination of critical parameters (MOI, DOT, and medium supplementation) as key factors in optimizing the upstream process. This finding impacts substantially upstream stage efficiency and economy, which could be useful for future scaling up to the commercial manufacturing scale.

Woody hosts are notoriously resistant to genetic transformation. Traditional methods, such as Agrobacterium-mediated transformation, are often inefficient, and this limitation extends to delivering infectious clones to woody plants. Dodder species (Cuscuta spp.) are holoparasitic plants that can establish direct connections with the vascular tissue of the parasitized plants, allowing them to facilitate virus transmission between unrelated botanical species. We demonstrated that a novel dodder-based approach achieved superior transmission in Rubus spp. compared to direct agroinoculation. The transmission rates for systemic blackberry chlorotic ringspot virus transmission increased from 9 % to 73 %, whereas the transmission of the phloem-limited blackberry yellow vein associated virus rose from 0 % to 46 %. This novel method expands the toolbox available to plant biologists to study virus-host interactions in woody plants.

Human adenoviruses (HAdV) are frequently excreted in large quantities and persist for extended periods in the environment, posing a significant health risk related to waterborne gastroenteritis. The objective of this study was to evaluate an adsorption-elution method using a negatively charged nitrocellulose membrane for its effectiveness in recovering HAdV from three different types of water (mineral water, tap water and well water). The detection of HAdV was carried out using real-time PCR. For this purpose, sterilized water samples were spiked with HAdV-infected stool and filtered through an electronegative membranes coated with MgCl₂ to retain viral particles. Subsequently, the viruses were eluted from the filters using sodium hydroxide and concentrated through two centrifugation cycles. Viral nucleic acids were then extracted and detected by real time PCR. Regarding HAdV recovery, the method's efficiency varied depending on the type of analyzed water. However, this method demonstrated a consistent performance, providing reliable results across different water samples, whether from mineral water, tap water or well water. This consistency in viral recovery is crucial to ensuring the accuracy and reliability of virological analyses in various aquatic environments.

A robust serological surveillance system for zoonotic pathogens is imperative for both early detection and advancing knowledge of emerging diseases. A statistical analysis plan that is aligned to research and epidemiological goals requires a purposeful choice among alternative methods for differentiating seronegative from seropositive samples, estimating seroprevalence, and estimating risk factors associated with seropositivity. The common standard deviation-based cutoff (e.g., 3sd) approach is simple to implement and understand, but fails to appropriately propagate uncertainty in serostatus assignments to any risk factor analysis. Methods such as Gaussian mixture models, which jointly estimate serostatus, risk factors, and their uncertainty, can alleviate the dichotomy created by the cutoff approach. Yet, because of a lack of empirical guidance of method performance, it remains difficult to choose a robust analysis method for a given serological dataset. Here we examine the performance of both cutoff and clustering approaches using simulated datasets that represent the epidemiological, biological, and immunological data generation process. We focus on understudied pathogens for which validated serological assays do not exist, as is common in emerging viruses in wildlife. We quantify coverage (the proportion of time 95 % confidence intervals contain the true value) and bias (systematic differences between true values and model point estimates) of model estimates for individual serostatus assignments, population seroprevalence, and regression coefficients for serostatus risk factors. In nearly all scenarios, Bayesian mixture models provide the highest coverage and lowest bias. Only with very low seroprevalence (∼ < 3 %) and large differences in signal between seronegative and seropositive individuals will a cutoff provide low bias and near-nominal coverage. Given poor coverage of risk factor regression coefficients, we advise against using a cutoff approach for quantifying determinants of seropositivity.

The Zika virus (ZIKV) - Orthoflavivirus zikaense - epidemic and its association with severe neurological disorders have created an urgent need to understand the disease pathogenesis and identify potential therapeutic targets. In previous investigations, we have shown that oxidative stress is associated with the pathogenesis of ZIKV infection in vitro and in vivo, and that silymarin has anti-ZIKV action in vitro. Here, we characterised the antioxidant and antiviral effects of silymarin against ZIKV infection in an animal model. We observed an increase in the levels of biomarkers of oxidative damage and in antioxidant enzyme activities in the livers of ZIKV-infected C57BL/6 mice. However, these effects were reversed in ZIKV-infected animals that were treated with silymarin. Furthermore, silymarin reduced the viral load in the livers of animals. Next, by in vitro studies, we confirmed that the anti-ZIKV action of silymarin is independent of its antioxidant activity. This work reinforces the potential use of silymarin against Zika fever.

Golden Syrian hamsters are an often-overlooked model in behavioral testing. While previously utilized for research examining circadian rhythms and mammalian reproduction, they are less common than murine models in both infectious disease and behavioral studies. However, coronavirus disease-19 (COVID-19) quickly pushed hamster modeling to the forefront due to its myriad of advantages over mice in recapitulating human pathology and transmission. At least 10 % of COVID-19 survivors suffer from post-acute sequelae of COVID-19 (PASC), a collection of some 200 sequelae with neurologic sequelae (neuro-PASC) presenting with potentially debilitating symptomology. This presents a clear need for a small animal model that recapitulates human disease with the ability to assess any potential long term neurological changes. We adapted and optimized a panel of behavioral tests from previously accepted murine models utilizing the golden Syrian hamster model for use within biocontainment facilities. Our panel includes grip strength, Porsolt forced swim, and novel object recognition testing to measure muscle fatigue or weakness, depression, and memory loss or cognitive impairment, respectively. Apart from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), this panel of tests is applicable to other pathogens that cause neurologic sequelae, such as Nipah or eastern equine encephalitis viruses, or any other model systems that require the use of hamsters. In this manuscript, we detail the methods for each of these three behavioral tests, how to interpret and analyze the resulting data, and emphasize additional factors for consideration. We also provide baseline data for both male and female golden Syrian hamsters.

The assembly of replication factors into functional complexes is crucial for the initiation of viral genome replication and processing of nascent viral DNA. Binding to viral DNA and interaction of protein domains presumably guide compartmentalization of replication factors. The phase separation due to hydrophilicity and hydrophobicity of components may also contribute to the assembling process. However, phase separation effects are poorly investigated in the infection cycle of baculoviruses, large DNA viruses infecting Diptera, Hymenoptera, and Lepidoptera insects. Herein, we describe an investigation on a possible role of phase separation in the assembly of nuclear replication factories in Spodoptera frugiperda Sf9 cells infected with the Autographa californica multiple nucleopolyhedrovirus (AcMNPV). The inhibitory effect of 1,6-Hexanediol on the translocation of a viral DNA binding protein (DBP) to the replicative centers has revealed the involvement of liquid phases separation in the assembly of these centers. DBP is a structural component of the virogenic stroma, a sub-nuclear membrane-less compartment involved in viral DNA replication and the production of nucleocapsids. This sub-nuclear structure is presumably assembled via a biomolecular condensation mechanism.

This study assessed the relative clinical sensitivity and specificity for cervical precancer of the Roche cobas HPV test when processed using the Roche cobas 6800 system using the Roche cobas 4800 HPV test as comparator. Intra- and inter-laboratory reproducibility were evaluated as well. The cobas HPV test run on the cobas 6800 platform demonstrated a relative clinical sensitivity of 1.00 for histologically confirmed CIN2 + lesions in woman aged 30 years or older, with a relative clinical specificity of 1.001 (p for non-inferior accuracy < 0.0001). The intra- and inter-laboratory reproducibility were 99.6 % and 99.8 % respectively. The study is the third to show that cobas HPV testing on the 6800 platform consistently demonstrates a relative clinical sensitivity of ≥ 0.95 and a relative clinical specificity of ≥ 0.98 for CIN2 + . This would qualify it, according to a recently published criteria, as a second-generation comparator assay.

Accurate diagnosis of dengue in the acute phase is crucial for proper patient management, reducing mortality rates, and enabling public health authorities to implement effective vector control measures. Our study, conducted during the 2024 dengue epidemic in Brazil, compared the results of a rapid dengue NS1 antigen detection assay with dengue RT-PCR using 300 serum specimens. Ninety-five percent of the patients reported symptoms consistent with dengue infection. We observed substantial agreement between the two tests (kappa = 0.8). The rapid NS1 test demonstrated 87% sensitivity and 92.7% specificity, using dengue RT-PCR as the gold standard. Our findings confirm that the rapid NS1 antigen detection test can be reliably used during the acute phase of dengue infection to provide an accurate diagnosis.

The tissue culture infectious dose 50 (TCID) end-point dilution assay is the gold-standard assay to titer viruses with negligible or ambiguous cytopathic effects. The assay's specificity is improved when followed by an Enzyme-Linked Immunosorbent Assay (ELISA) to detect viral antigens. Cells infected with mosquito-borne orthoflavi- and alphaviruses are fixed after TCID, prior to ELISA, using paraformaldehyde (PFA) or acetone. While 4 % PFA has been shown to effectively inactivate these viruses for safe handling in low biocontainment conditions, equivalent studies have not been reported for standard acetone fixation methods (20 % acetone for 24 hours at 4°C). This study evaluated the inactivation efficacy of acetone on orthoflavi- and alphaviruses using dengue virus (DENV) and Ross River virus (RRV), as exemplar viruses from each genus, respectively. We show that 50 % acetone and 4 % PFA fully inactivate DENV and RRV, but 20 % acetone does not reduce the infectivity of these viruses. Importantly, ELISA-based detection of DENV- and RRV-infected cells fixed with 50 % acetone was effective, with calculated titres comparable to cells treated with 20 % acetone. Together, our results inform a fixation method for titrating orthoflavi- and alphavirus samples by TCID/ELISA, ensuring the safe handling and processing of these viruses under low biocontainment conditions.

We assessed the viability of aerosolized human betacoronavirus OC43 (HCoV-OC43; ATCC VR-1558), human rhinovirus-14 (RV-14; ATCC VR-284) and feline calicivirus (FCV; ATCC VR-782) as representative enveloped and non-enveloped respiratory and enteric viruses of mammals in indoor air under ambient conditions (relative humidity 50 ± 10 % and air temperature 22 ± 2°C) using a room-sized (25 m; 900 ft) aerobiology chamber. All virus suspensions contained a soil load to simulate the presence of body fluids and they were separately aerosolized into the chamber using a six-jet Collison nebulizer. A muffin fan was used to uniformly mix the air inside the chamber and to keep the aerosols airborne. A slit sampler with Petri plates containing 3 % (wt./vol) gelatin was used to collect the air samples. The gelatin was liquefied in an incubator and assayed for infectious virus as plaque-forming units (PFU). The rates of biological decay of HCoV-OC43, RV-14 and FCV were 0.0052 ± 0.00026, 0.0034 ± 0.0027 and 0.0081 ± 0.0031 (as log PFU/m/min), respectively. We also assessed a HEPA filter-based stand-alone air purifier against the experimentally aerosolized viruses and the device could demonstrate > 3-log reductions in the viability of the three viruses in 46, 62 and 41 minutes, respectively. Therefore, we can now investigate the stability of mammalian viruses in indoor air as well as air decontamination technologies against them under field-relevant conditions.

Infant mortality remains a major global concern. Sudden unexpected death in infancy (SUDI) is reported globally and an infant mortality rate of 23.129 per 1 000 live births has been reported in the Western Cape, South Africa, in 2024. Infections are often confirmed in SUDI cases admitted to the Tygerberg Medico-legal Mortuary in Cape Town, but molecular diversity in respiratory viruses is underreported. A total of 162 previously confirmed polymerase chain reaction (PCR)-positive trachea and / or lung samples from SUDI cases collected between 2015 and 2019 were retested for either rhinovirus or human respiratory syncytial virus (RSV). Sixty-four samples were positive for rhinovirus and 15 for RSV. Results from 5 of all positive samples were outside the PCR assay amplification limits determined by the cycle threshold (Ct) value and were excluded. Another 4 samples did not amplify, and the remaining 70 underwent subsequent sequencing, but successful sequences could only be obtained in 53 samples. All three rhinovirus (A, B and C) genotypes were identified, with RV-A most prevalent, followed by RV-C and RV-B. RSV-A and RSV-B were detected equally, and after amino acid alignment, 20 amino acid duplication and nine substitutions were found that confirmed two RSV-BA9 genotypes. This study describes the molecular and phylogenetic characterisation of specific respiratory viruses in SUDI cases in South Africa. However, the rapid decline in viral viability in post-mortem samples does not allow correlation between viral genotypes and cause of death or disease severity. Future prospective studies should therefore investigate temporality and associations between specific viral strains and clinical disease severity and mortality.

Human enterovirus D68 (EV-D68) has been associated with an increase in mild-to-severe pediatric respiratory diseases worldwide. The rate of circulation of this virus is largely underestimated in the population and genetic evolutionary data are usually available only for partial sequences. To achieve a timely genomic surveillance, a reliable, high-throughput EV-68 sequencing assay is required. Here we report an improved high-throughput EV-D68 whole-genome sequencing assay performed directly on clinical samples that is suitable for short-read sequencing platforms. Between June and December 2022, a total 37 (1.9 %) respiratory samples were EV-D68 positive and together with 52 additional samples with a median cycle of quantification (Cq) of 28.3, ranging from 18 to 36.8 Cq were included in the validation analyses. Overall, all the primers had good performance and no mismatches were detected in more than 85 % of sequences (932 whole-genome dataset). Using a cut-off of Cq < 32 in at least 85.5 % of samples a whole-genome or partial genome was obtained, confirming an acceptable positive sequencing rate for the designed method. A total of 65 whole-genome sequences were obtained and have a mean coverage of 98.4 % across the genome, with a median depth of 6158x (range 2815x-7560x). Based on the obtained data, this method is cost effective resulting in an easy-to-perform protocol helpful for tracing the evolution of EV-D68 in protein different from VP1. EV-D68 could become a significant pathogen for public health in the next future, and thus this protocol for whole genome sequencing could help clinical and molecular virologists to be ready for molecular epidemiology surveillance.

To detect viruses such as hepatitis A virus (HAV) and human norovirus (HuNoV) in foods, RT-qPCR or other molecular methods are used, which cannot distinguish between infectious and non-infectious virions. Samples can be pretreated to limit detection to intact and presumably infectious virions. We compared propidium monoazide (PMA or PMAxx), platinum (IV) chloride (PtCl), magnetic silica beads and centrifugal filter using HAV or HuNoV inactivated by heat, pulsed light, or sodium hypochlorite (NaOCl). PMAxx completely or nearly eliminated (3.96 ± 1.24 log gc) the RT-qPCR signal of HAV inactivated at 100°C for 10 min. Pretreatments could not reduce significantly RT-qPCR signal of HAV after pulsed light (0.74 ± 0.36 log gc) and NaOCl (0.24 ± 0.14 log gc) inactivation. Enzymatic treatments did not improve the results obtained with PMAxx. The exudate of raspberry, strawberry or oyster used as food matrices needed dilution by at least tenfold for PMAxx to to yield results comparable to virions without a food matrix. Overall, PMAxx shows good potential to discriminate between infectious and non-infectious despite some remaining limitations.