ABSTRACT
The emergence of SARS-CoV-2 in late 2019 initiated a global pandemic, which led to a need for effective therapeutics and diagnostic tools, including virus-specific antibodies. Here, we investigate different antigen preparations to produce SARS-CoV-2-specific and virus-neutralizing antibodies in chickens (n = 3/antigen) and rabbits (n = 2/antigen), exploring, in particular, egg yolk for large-scale production of immunoglobulin Y (IgY). Reactivity profiles of IgY preparations from chicken sera and yolk and rabbit sera were tested in parallel. We compared three types of antigens based on ancestral SARS-CoV-2: an inactivated whole-virus preparation, an S1 spike-protein subunit (S1 antigen) and a receptor-binding domain (RBD antigen, amino acids 319-519) coated on lumazine synthase (LS) particles using SpyCather/SpyTag technology. The RBD antigen proved to be the most efficient immunogen, and the resulting chicken IgY antibodies derived from serum or yolk, displayed strong reactivity with ELISA and indirect immunofluorescence and broad neutralizing activity against SARS-CoV-2 variants, including Omicron BA.1 and BA.5. Preliminary in vivo studies using RBD-lumazine synthase yolk preparations in a hamster model showed that local application was well tolerated and not harmful. However, despite the in vitro neutralizing capacity, this antibody preparation did not show protective effect. Further studies on galenic properties seem to be necessary. The RBD-lumazine antigen proved to be suitable for producing SARS-CoV-2 specific antibodies that can be applied to such therapeutic approaches and as reference reagents for SARS-CoV-2 diagnostics, including virus neutralization assays.
Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , COVID-19 , Chickens , Immunoglobulins , SARS-CoV-2 , Animals , SARS-CoV-2/immunology , Immunoglobulins/immunology , Chickens/immunology , Antibodies, Neutralizing/immunology , Antibodies, Viral/immunology , Rabbits , COVID-19/immunology , COVID-19/virology , Spike Glycoprotein, Coronavirus/immunology , Humans , Neutralization TestsABSTRACT
Little is known about the frequency of Zika virus (ZIKV) infections in Sudan. The aim of this study was to obtain data on the prevalence of ZIKV infections and the immunity of the population in the country. To this end, 198 sera obtained between December 2012 and January 2013 in different regions in Sudan were examined for neutralizing antibodies against ZIKV, dengue virus (DENV), and yellow fever virus (YFV). The sera were non-randomly selected. The neutralization titers were compared with each other and with the WHO 1st International Standard for anti-Asian lineage Zika virus antibody. Twenty-six sera neutralized ZIKV. One-third of these sera had higher neutralization titers against ZIKV than against DENV-2 and -3. Two sera showed higher neutralization titers than the WHO standard for ZIKV antibodies. These data suggest occasional ZIKV infections in Sudan. The low percentage of sera in this cohort that neutralized ZIKV indicates that, in the study period, the population was susceptible to ZIKV infection.
Subject(s)
Antibodies, Neutralizing , Antibodies, Viral , Zika Virus Infection , Zika Virus , Sudan/epidemiology , Humans , Zika Virus Infection/epidemiology , Zika Virus Infection/immunology , Zika Virus Infection/blood , Antibodies, Viral/blood , Zika Virus/immunology , Antibodies, Neutralizing/blood , Antibodies, Neutralizing/immunology , Adult , Female , Male , Middle Aged , Young Adult , Adolescent , Neutralization Tests , Dengue Virus/immunology , Child , Yellow fever virus/immunology , Seroepidemiologic Studies , Child, Preschool , Aged , PrevalenceABSTRACT
This article presents a demonstration of the improved performance of an X-ray free-electron laser (FEL) using the optical klystron mechanism and helical undulator configuration, in comparison with the common planar undulator configuration without optical klystron. The demonstration was carried out at Athos, the soft X-ray beamline of SwissFEL. Athos has variable-polarization undulators, and small magnetic chicanes placed between every two undulators to fully exploit the optical klystron. It was found that, for wavelengths of 1.24â nm and 3.10â nm, the required length to achieve FEL saturation is reduced by about 35% when using both the optical klystron and helical undulators, with each effect accounting for about half of the improvement. Moreover, it is shown that a helical undulator configuration provides a 20% to 50% higher pulse energy than planar undulators. This work represents an important step towards more compact and high-power FELs, rendering this key technology more efficient, affordable and accessible to the scientific community.
ABSTRACT
The aim of this study was to analyse the hygienic suitability of wood often used in animal husbandry. To this end, the inactivation of viruses (Enterovirus E as a surrogate for non-enveloped viruses and Newcastle disease virus as a surrogate for enveloped viruses) on germ carriers consisting of various types of wood was studied over an extended period to assess the biosafety of wood as an agricultural building material. The study was designed to assess the intrinsic biocidal activity of the wood itself, without the use of a disinfectant. The laboratory tests were based on German test guidelines and current European standards. Five different types of wood germ carriers, i.e., spruce (Picea abies), pine (Pinus sylvestris), poplar (Populus sp.), beech (Fagus sylvatica) and Douglas fir (Pseudotsuga menziesii), as well as stainless-steel carriers, were inoculated with enveloped and non-enveloped viruses and stored for up to four months, and the remaining infectivity of the viruses was continuously assessed. The results showed that intact, finely sawn timber with a low depth of roughness had an inactivating effect on the viruses up to 7.5 decadal logarithmic levels. For the non-enveloped virus, inactivation was fastest on Douglas fir wood, with the target reduction for effective inactivation (reduction by factor 4.0 log10) being achieved after two weeks, and for the enveloped virus on pine wood, it was already achieved from the day of drying. The hygienic effects of the wood carriers may be due to their hygroscopic properties and wood constituents. These effects offer potential for further investigation, including tests with other wood species rich in extractives.
Subject(s)
Animal Husbandry , Wood , Wood/virology , Animals , Animal Husbandry/methods , Virus InactivationABSTRACT
The aim of this study was to test the inactivation of viruses on germ carriers of different types of wood using a disinfectant in order to assess the biosafety of wood as a building material in animal husbandry. The laboratory disinfectant efficacy tests were based on German testing guidelines and current European standards. Five different types of wood germ carriers, i.e., spruce (Picea abies), pine (Pinus sylvestris), poplar (Populus sp.), beech (Fagus sylvatica) and Douglas fir (Pseudotsuga menziesii), were inoculated with enveloped or non-enveloped viruses and then treated with one of three different disinfectants. The results revealed that intact, fine-sawn timber with a low roughness depth can be effectively inactivated. Peracetic acid proved to be the most effective disinfectant across all tests. Regardless of the pathogen and the type of wood, a concentration of 0.1% of the pure substance at a temperature of 10 °C and an exposure time of one hour can be recommended. At a temperature of -10 °C, a concentration of 0.75% is recommended. The basic chemicals formic acid and glutaraldehyde demonstrated only limited effectiveness overall. The synergistic effects of various wood components on the inactivation of viruses offer potential for further investigation. Disinfectant tests should also be conclusively verified in field trials to ensure that the results from standardised laboratory tests can be transferred to real stable conditions.
ABSTRACT
A new method for time-resolved X-ray absorption near edge structure (XANES) spectroscopy that enables faster data acquisition and requires smaller sample quantities for high-quality data, thus allowing the analysis of more samples in a shorter time is introduced. The method uses large bandwidth free electron laser pulses to measure laser-excited XANES spectra in transmission mode. A beam-splitting grating configuration allows simultaneous measurements of the spectra of the incoming X-ray Free Electron Laser (XFEL) pulses and transmission XANES, which is crucial for compensating the pulse-dependent intensity and spectrum fluctuations due to the self-amplified spontaneous emission operation. The implementation of this new methodology is applied on a liquid solution of ammonium iron(III) oxalate jet and is compared to previous results, showing great improvements in the speed of acquisition and spectral resolution, and the ability to measure a large 2-D spectral-time map quickly.
ABSTRACT
We present the generation of x-ray pulses with average pulse energies up to one millijoule and rms pulse durations down to the femtosecond level. We have produced these intense and short pulses by employing the fresh-slice multistage amplification scheme with a transversely tilted electron beam in a free-electron laser. In this scheme, a short pulse is produced in the first stage and later amplified by fresh parts of the electron bunch in up to a total of four stages of amplification. Our implementation is efficient, since practically the full electron beam contributes to produce the x-ray pulse. Our implementation is also compact, utilizing only 32 m of undulator. The demonstration was done at Athos, the soft x-ray beamline of SwissFEL, which was designed with high flexibility to take full advantage of the multistage amplification scheme. It opens the door for scientific opportunities following ultrafast dynamics using nonlinear x-ray spectroscopy techniques or avoiding electronic damage when capturing structures with a single intense pulse via single-particle imaging.
ABSTRACT
Viral disinfection is important for medical facilities, the food industry, and the veterinary field, especially in terms of controlling virus outbreaks. Therefore, standardized methods and activity levels are available for these areas. Usually, disinfectants used in these areas are characterized by their activity against test organisms (i.e., viruses, bacteria, and/or yeasts). This activity is usually determined using a suspension test in which the test organism is incubated with the respective disinfectant in solution to assess its bactericidal, yeasticidal, or virucidal activity. In addition, carrier methods that more closely reflect real-world applications have been developed, in which microorganisms are applied to the surface of a carrier (e.g., stainless steel frosted glass, or polyvinyl chloride (PVC)) and then dried. However, to date, no standardized methods have become available for addressing genetically modified vectors or disinfection-resistant oncolytic viruses such as the H1-parvovirus. Particularly, such non-enveloped viruses, which are highly resistant to disinfectants, are not taken into account in European standards. This article proposes a new activity claim known as "virucidal activity PLUS", summarizes the available methods for evaluating the virucidal activity of chemical disinfectants against genetically modified organisms (GMOs) using current European standards, including the activity against highly resistant parvoviridae such as the adeno-associated virus (AAV), and provides guidance on the selection of disinfectants for pharmaceutical manufacturers, laboratories, and clinical users.
Subject(s)
Disinfectants , Parvoviridae Infections , Parvovirus , Viruses , Humans , Disinfectants/pharmacology , Disinfection/methods , Viruses/geneticsABSTRACT
X-ray free-electron lasers (FELs) are state-of-the-art scientific tools capable to study matter on the scale of atomic processes. Since the initial operation of X-ray FELs more than a decade ago, several facilities with upgraded performance have been put in operation. Here we present the first lasing results of Athos, the soft X-ray FEL beamline of SwissFEL at the Paul Scherrer Institute in Switzerland. Athos features an undulator layout based on short APPLE-X modules providing full polarisation control, interleaved with small magnetic chicanes. This versatile configuration allows for many operational modes, giving control over many FEL properties. We show, for example, a 35% reduction of the required undulator length to achieve FEL saturation with respect to standard undulator configurations. We also demonstrate the generation of more powerful pulses than the ones obtained in typical undulators. Athos represents a fundamental step forward in the design of FEL facilities, creating opportunities in FEL-based sciences.
ABSTRACT
N6-methyladenosine (m6A) is one of the most abundant modifications of cellular RNA, where it serves various functions. m6A methylation of many viral RNA species has also been described; however, little is known about the m6A epitranscriptome of haemorrhagic fever-causing viruses like Ebola virus (EBOV). Here, we analysed the importance of the methyltransferase METTL3 for the life cycle of this virus. We found that METTL3 interacts with the EBOV nucleoprotein and the transcriptional activator VP30 to support viral RNA synthesis, and that METTL3 is recruited into EBOV inclusions bodies, where viral RNA synthesis occurs. Analysis of the m6A methylation pattern of EBOV mRNAs showed that they are methylated by METTL3. Further studies revealed that METTL3 interaction with the viral nucleoprotein, as well as its importance for RNA synthesis and protein expression, is also observed for other haemorrhagic fever viruses such as Junín virus (JUNV) and Crimean-Congo haemorrhagic fever virus (CCHFV). The negative effects on viral RNA synthesis due to loss of m6A methylation are independent of innate immune sensing, as METTL3 knockout did not affect type I interferon induction in response to viral RNA synthesis or infection. Our results suggest a novel function for m6A that is conserved among diverse haemorrhagic fever-causing viruses (i.e. EBOV, JUNV and CCHFV), making METTL3 a promising target for broadly-acting antivirals.
Subject(s)
Dengue Virus , Ebolavirus , Hemorrhagic Fever Virus, Crimean-Congo , Hemorrhagic Fever, Ebola , Humans , Hemorrhagic Fever Virus, Crimean-Congo/genetics , Ebolavirus/genetics , RNA, Viral/genetics , RNA, Viral/metabolism , Dengue Virus/genetics , Nucleoproteins , Methyltransferases/geneticsABSTRACT
The Paul Scherrer Institute is implementing laser-based seeding in the soft X-ray beamline (Athos) of its free-electron laser, SwissFEL, to enhance the temporal and spectral properties of the delivered photon pulses. This technique requires, among other components, two identical modulators for coupling the electron beam with an external laser with a wavelength range between 260 and 1600â nm. The design, magnetic measurements results, alignment, operation and also details of the novel and exotic magnetic configuration of the prototype are described.
ABSTRACT
La Crosse virus, responsible for pediatric encephalitis in the United States, and Schmallenberg virus, a highly teratogenic veterinary virus in Europe, belong to the large Orthobunyavirus genus of zoonotic arthropod-borne pathogens distributed worldwide. Viruses in this under-studied genus cause CNS infections or fever with debilitating arthralgia/myalgia syndromes, with no effective treatment. The main surface antigen, glycoprotein Gc (â¼1,000 residues), has a variable N-terminal half (GcS) targeted by the patients' antibody response and a conserved C-terminal moiety (GcF) responsible for membrane fusion during cell entry. Here, we report the X-ray structure of post-fusion La Crosse and Schmallenberg virus GcF, revealing the molecular determinants for hairpin formation and trimerization required to drive membrane fusion. We further experimentally confirm the role of residues in the fusion loops and in a vestigial endoplasmic reticulum (ER) translocation sequence at the GcS-GcF junction. The resulting knowledge provides essential molecular underpinnings for future development of potential therapeutic treatments and vaccines.
Subject(s)
La Crosse virus , Orthobunyavirus , Humans , Child , Orthobunyavirus/genetics , Orthobunyavirus/chemistry , Membrane Glycoproteins , Membrane Fusion , GlycoproteinsABSTRACT
'Staggering disease' is a neurological disease entity considered a threat to European domestic cats (Felis catus) for almost five decades. However, its aetiology has remained obscure. Rustrela virus (RusV), a relative of rubella virus, has recently been shown to be associated with encephalitis in a broad range of mammalian hosts. Here, we report the detection of RusV RNA and antigen by metagenomic sequencing, RT-qPCR, in-situ hybridization and immunohistochemistry in brain tissues of 27 out of 29 cats with non-suppurative meningoencephalomyelitis and clinical signs compatible with'staggering disease' from Sweden, Austria, and Germany, but not in non-affected control cats. Screening of possible reservoir hosts in Sweden revealed RusV infection in wood mice (Apodemus sylvaticus). Our work indicates that RusV is the long-sought cause of feline 'staggering disease'. Given its reported broad host spectrum and considerable geographic range, RusV may be the aetiological agent of neuropathologies in further mammals, possibly even including humans.
Subject(s)
Encephalomyelitis , Humans , Animals , Cats , Mice , Causality , Sweden , Austria , Germany , MammalsABSTRACT
X-ray free-electron lasers (FELs) deliver ultrabright X-ray pulses, but not the sequences of phase-coherent pulses required for time-domain interferometry and control of quantum states. For conventional split-and-delay schemes to produce such sequences, the challenge stems from extreme stability requirements when splitting Ångstrom wavelength beams, where the tiniest path-length differences introduce phase jitter. We describe an FEL mode based on selective electron-bunch degradation and transverse beam shaping in the accelerator, combined with a self-seeded photon emission scheme. Instead of splitting the photon pulses after their generation by the FEL, we split the electron bunch in the accelerator, prior to photon generation, to obtain phase-locked X-ray pulses with subfemtosecond duration. Time-domain interferometry becomes possible, enabling the concomitant program of classical and quantum optics experiments with X-rays. The scheme leads to scientific benefits of cutting-edge FELs with attosecond and/or high-repetition rate capabilities, ranging from the X-ray analog of Fourier transform infrared spectroscopy to damage-free measurements.
ABSTRACT
Crimean-Congo haemorrhagic fever virus (CCHFV) continues to cause new human cases in Iberia while its spatial distribution and ecological determinants remain unknown. The virus remains active in a silent tick-animal cycle to which animals contribute maintaining the tick populations and the virus itself. Wild ungulates, in particular red deer, are essential hosts for Hyalomma ticks in Iberia, which are the principal competent vector of CCHFV. Red deer could be an excellent model to understand the ecological determinants of CCHFV as well as to predict infection risks for humans because it is large, gregarious, abundant and the principal host for Hyalomma lusitanicum. We designed a cross-sectional study, analysed the presence of CCHFV antibodies in 1444 deer from 82 populations, and statistically modelled exposure risk with host and environmental predictors. The best-fitted statistical model was projected for peninsular Spain to map infection risks. Fifty out of 82 deer populations were seropositive, with individual population prevalence as high as 88%. The highest prevalence of exposure to CCHFV occurred in the southwest of the Iberian Peninsula. Climate and ungulate abundance were the most influential predictors of the risk of exposure to the virus. The highest risk regions were those where H. lusitanicum is most abundant. Eight of the nine primary human cases occurred in or bordering these regions, demonstrating that the model predicts human infection risk accurately. A recent human case of CCHF occurred in northwestern Spain, a region that the model predicted as low risk, pointing out that it needs improvement to capture all determinants of the CCHFV infection risk. In this study, we have been able to identify the main ecological determinants of CCHFV, and we have also managed to create an accurate model to assess the risk of CCHFV infection.
Subject(s)
Deer , Hemorrhagic Fever Virus, Crimean-Congo , Hemorrhagic Fever, Crimean , Ixodidae , Ticks , Animals , Cross-Sectional Studies , Hemorrhagic Fever, Crimean/epidemiology , Hemorrhagic Fever, Crimean/veterinary , HumansABSTRACT
Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables essentially radiation-damage-free macromolecular structure determination using microcrystals that are too small for synchrotron studies. However, SFX experiments often require large amounts of sample in order to collect highly redundant data where some of the many stochastic errors can be averaged out to determine accurate structure-factor amplitudes. In this work, the capability of the Swiss X-ray free-electron laser (SwissFEL) was used to generate large-bandwidth X-ray pulses [Δλ/λ = 2.2% full width at half-maximum (FWHM)], which were applied in SFX with the aim of improving the partiality of Bragg spots and thus decreasing sample consumption while maintaining the data quality. Sensitive data-quality indicators such as anomalous signal from native thaumatin micro-crystals and de novo phasing results were used to quantify the benefits of using pink X-ray pulses to obtain accurate structure-factor amplitudes. Compared with data measured using the same setup but using X-ray pulses with typical quasi-monochromatic XFEL bandwidth (Δλ/λ = 0.17% FWHM), up to fourfold reduction in the number of indexed diffraction patterns required to obtain similar data quality was achieved. This novel approach, pink-beam SFX, facilitates the yet underutilized de novo structure determination of challenging proteins at XFELs, thereby opening the door to more scientific breakthroughs.
ABSTRACT
Background: Technical protection measures for laboratory activities involving biological agents include biological safety cabinets (BSC) that may be contaminated. In the case of diagnostic activities with SARS-CoV-2, this may also affect BSC that are operated at protection level 2; therefore, decontamination of all contaminated surfaces of the BSC may be required. In addition to fumigation with hydrogen peroxide (H2O2), dry fogging of H2O2-stabilized peroxyacetic acid (PAA) represents another alternative to fumigation with formalin. However, to prove their efficacy, these alternatives need to be validated for each model of BSC. Methods: The validation study was performed on 4 different BSCs of Class II A2 using the "Mini Dry Fog" system. Results: An aerosol concentration of 0.03% PAA and 0.15% H2O2 during a 30 min exposure was sufficient to inactivate SARS-CoV-2. Effective concentrations of 1.0% PAA and 5% H2O2 were required to decontaminate the custom-prepared biological indicators loaded with spores of G. stearothermophilus and deployed at 9 different positions in the BSC. Commercial spore carriers were easier to inactivate by a factor of 4, which corresponded to a reduction of 106 in all localizations. Conclusions: Dry fogging with PAA is an inexpensive, robust, and highly effective decontamination method for BSCs for enveloped viruses such as SARS-CoV-2. The good material compatibility, lack of a requirement for neutralization, low pH - which increases the range of efficacy compared to H2O2 fumigation - the significantly shorter processing time, and the lower costs argue in favor of this method.
ABSTRACT
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a severe adverse effect of ChAdOx1 nCoV-19 COVID-19 vaccine (Vaxzevria) and Janssen Ad26.COV2.S COVID-19 vaccine, and it is associated with unusual thrombosis. VITT is caused by anti-platelet factor 4 (PF4) antibodies activating platelets through their FcγRIIa receptors. Antibodies that activate platelets through FcγRIIa receptors have also been identified in patients with COVID-19. These findings raise concern that vaccination-induced antibodies against anti-SARS-CoV-2 spike protein cause thrombosis by cross-reacting with PF4. Immunogenic epitopes of PF4 and SARS-CoV-2 spike protein were compared using in silico prediction tools and 3D modeling. The SARS-CoV-2 spike protein and PF4 share at least 1 similar epitope. Reactivity of purified anti-PF4 antibodies from patients with VITT was tested against recombinant SARS-CoV-2 spike protein. However, none of the affinity-purified anti-PF4 antibodies from 14 patients with VITT cross-reacted with SARS-CoV-2 spike protein. Sera from 222 polymerase chain reaction-confirmed patients with COVID-19 from 5 European centers were tested by PF4-heparin enzyme-linked immunosorbent assays and PF4-dependent platelet activation assays. We found anti-PF4 antibodies in sera from 19 (8.6%) of 222 patients with COVID-19. However, only 4 showed weak to moderate platelet activation in the presence of PF4, and none of those patients developed thrombotic complications. Among 10 (4.5%) of 222 patients who had COVID-19 with thrombosis, none showed PF4-dependent platelet-activating antibodies. In conclusion, antibodies against PF4 induced by vaccination do not cross-react with the SARS-CoV-2 spike protein, indicating that the intended vaccine-induced immune response against SARS-CoV-2 spike protein is not the trigger of VITT. PF4-reactive antibodies found in patients with COVID-19 in this study were not associated with thrombotic complications.
Subject(s)
Antibodies/adverse effects , COVID-19 Vaccines/adverse effects , Cross Reactions/immunology , Platelet Factor 4/immunology , Purpura, Thrombocytopenic, Idiopathic/etiology , Purpura, Thrombocytopenic, Idiopathic/immunology , Spike Glycoprotein, Coronavirus/immunology , Adult , Aged , Aged, 80 and over , Blood Platelets/immunology , COVID-19/immunology , Cohort Studies , Epitopes/immunology , Female , Heparin/metabolism , Humans , Immunoglobulin G/immunology , Male , Middle Aged , Protein Binding , Protein Domains , Purpura, Thrombocytopenic, Idiopathic/blood , Spike Glycoprotein, Coronavirus/chemistry , Young AdultABSTRACT
SARS CoV-2 antibody assays measure antibodies against the viral nucleoprotein (NP) or spike protein. The study examined if testing of antibodies against both antigens increases the diagnostic sensitivity. Sera (N=98) from infected individuals were tested with ELISAs based on the NP, receptor-binding domain (RBD), or both proteins. The AUROCs were 0.958 (NP), 0.991 (RBD), and 0.992 (NP/RBD). The RBD- and NP/RBD-based ELISAs showed better performance than the NP-based assay. Simultaneous testing for antibodies against NP and RBD increased the number of true and false positives. If maximum diagnostic sensitivity is required, the NP/RBD-based ELISA is preferable. Otherwise, the RBD-based ELISA is sufficient.
Subject(s)
Antibodies, Viral/blood , COVID-19 Testing/methods , COVID-19/blood , Enzyme-Linked Immunosorbent Assay/methods , Nucleoproteins/immunology , SARS-CoV-2/immunology , COVID-19/virology , Humans , Nucleoproteins/chemistry , Protein Domains , SARS-CoV-2/chemistryABSTRACT
BACKGROUND: Newcastle disease is a devastating disease in poultry caused by virulent Newcastle disease virus (NDV), a paramyxovirus endemic in many regions of the world despite intensive vaccination. Phylogenetic analyses reveal ongoing evolution of the predominant circulating genotype 2.VII, and the relevance of potential antigenic drift is under discussion. To investigate variation within neutralization-sensitive epitopes within the protein responsible for receptor binding, i.e. the Hemagglutinin-Neuraminidase (HN) spike protein, we were interested in establishing genotype-specific monoclonal antibodies (MAbs). METHODS: An HN-enriched fraction of a gradient-purified NDV genotype 2.VII was prepared and successfully employed to induce antibodies in BalbC mice that recognize conformationally intact sites reactive by haemagglutination inhibition (HI). For subsequent screening of mouse hybridoma cultures, an NDV-ELISA was established that utilizes Concanavalin A (ConA-ELISA) coupled glycoproteins proven to present conformation-dependent epitopes. RESULTS: Six out of nine selected MAbs were able to block receptor binding as demonstrated by HI activity. One MAb recognized an epitope only present in the homologue virus, while four other MAbs showed weak reactivity to selected other genotypes. On the other hand, one broadly cross-reacting MAb reacted with all genotypes tested and resembled the reactivity profile of genotype-specific polyclonal antibody preparations that point to minor antigenic differences between tested NDV genotpyes. CONCLUSIONS: These results point to the concurrent presence of variable and conserved epitopes within the HN molecule of NDV. The described protocol should help to generate MAbs against a variety of NDV strains and to enable in depth analysis of the antigenic profiles of different genotypes.