Chimeric virus-like particles presenting tumour-associated MUC1 epitope result in high titers of specific IgG antibodies in the presence of squalene oil-in-water adjuvant: towards safe cancer immunotherapy.

BACKGROUND: Immunotherapy is emerging as a powerful treatment approach for several types of cancers. Modulating the immune system to specifically target cancer cells while sparing healthy cells, is a very promising approach for safer therapies and increased survival of cancer patients. Tumour-associated antigens are favorable targets for cancer immunotherapy, as they are exclusively expressed by the cancer cells, minimizing the risk of an autoimmune reaction. The ability to initiate the activation of the immune system can be achieved by virus-like particles (VLPs) which are safe and potent delivery tools. VLP-based vaccines have evolved dramatically over the last few decades and showed great potential in preventing infectious diseases. Immunogenic potency of engineered VLPs as a platform for the development of effective therapeutic cancer vaccines has been studied extensively. This study involves recombinant VLPs presenting multiple copies of tumour-specific mucin 1 (MUC1) epitope as a potentially powerful tool for future immunotherapy. RESULTS: In this report VLPs based on the structural protein of Norovirus (NoV VP1) were genetically modified to present multiple copies of tumour-specific MUC1 epitope on their surface. Chimeric MUC1 particles were produced in the eukaryotic Leishmania tarentolae expression system and used in combination with squalene oil-in-water emulsion MF59 adjuvant to immunize BALB/c mice. Sera from vaccinated mice demonstrated high titers of IgG and IgM antibodies which were specifically recognizing MUC1 antigen. CONCLUSIONS: The obtained results show that immunization with recombinant chimeric NoV VP1- MUC1 VLPs result in high titers of MUC1 specific IgG antibodies and show great therapeutic potential as a platform to present tumour-associated antigens.

Immunization with Leishmania tarentolae-derived norovirus virus-like particles elicits high humoral response and stimulates the production of neutralizing antibodies.

BACKGROUND: Noroviruses are a major cause of epidemic and sporadic acute non-bacterial gastroenteritis worldwide. Unfortunately, the development of an effective norovirus vaccine has proven difficult and no prophylactic vaccine is currently available. Further research on norovirus vaccine development should be considered an absolute priority and novel vaccine candidates are needed. One of the recent approaches in safe vaccine development is the use of virus-like particles (VLPs). VLP-based vaccines show great immunogenic potential as they mimic the morphology and structure of viral particles without the presence of the virus genome. RESULTS: This study is the first report showing successful production of norovirus VLPs in the protozoan Leishmania tarentolae (L. tarentolae) expression system. Protozoan derived vaccine candidate is highly immunogenic and able to not only induce a strong immune response (antibody titer reached 104) but also stimulate the production of neutralizing antibodies confirmed by receptor blocking assay. Antibody titers able to reduce VLP binding to the receptor by > 50% (BT50) were observed for 1:5-1:320 serum dilutions. CONCLUSIONS: Norovirus VLPs produced in L. tarentolae could be relevant for the development of the norovirus vaccine.

Immunosensor Based on Long-Period Fiber Gratings for Detection of Viruses Causing Gastroenteritis.

Since the norovirus is the main cause of acute gastroenteritis all over the world, its fast detection is crucial in medical diagnostics. In this work, a rapid, sensitive, and selective optical fiber biosensor for the detection of norovirus virus-like particles (VLPs) is reported. The sensor is based on highly sensitive long-period fiber gratings (LPFGs) coated with antibodies against the main coat protein of the norovirus. Several modification methods were verified to obtain reliable immobilization of protein receptors on the LPFG surface. We were able to detect 1 ng/mL norovirus VLPs in a 40-min assay in a label-free manner. Thanks to the application of an optical fiber as the sensor, there is a possibility to increase the user's safety by separating the measurement point from the signal processing setup. Moreover, our sensor is small and light, and the proposed assay is straightforward. The designed LPFG-based biosensor could be applied in both fast norovirus detection and in vaccine testing.

Immunogenicity of Leishmania-derived hepatitis B small surface antigen particles exposing highly conserved E2 epitope of hepatitis C virus.

BACKGROUND: Hepatitis C virus (HCV) infection is a major health problem worldwide, affecting an estimated 2-3 % of human population. An HCV vaccine, however, remains unavailable. High viral diversity poses a challenge in developing a vaccine capable of eliciting a broad neutralizing antibody response against all HCV genotypes. The small surface antigen (sHBsAg) of hepatitis B virus (HBV) has the ability to form highly immunogenic subviral particles which are currently used as an efficient anti-HBV vaccine. It also represents an attractive antigen carrier for the delivery of foreign sequences. In the present study, we propose a bivalent vaccine candidate based on novel chimeric particles in which highly conserved epitope of HCV E2 glycoprotein (residues 412-425) was inserted into the hydrophilic loop of sHBsAg. RESULTS: The expression of chimeric protein was performed in an unconventional, Leishmania tarentolae expression system resulting in an assembly of particles which retained immunogenicity of both HCV epitope and sHBsAg protein. Direct transmission electron microscopy observation and immunogold staining confirmed the formation of spherical particles approximately 22 nm in diameter, and proper foreign epitope exposition. Furthermore, the sera of mice immunized with chimeric particles proved reactive not only to purified yeast-derived sHBsAg proteins but also HCV E2 412-425 synthetic peptide. Most importantly, they were also able to cross-react with E1E2 complexes from different HCV genotypes. CONCLUSIONS: For the first time, we confirmed successful assembly of chimeric sHBsAg virus-like particles (VLPs) in the L. tarentolae expression system which has the potential to produce high-yields of properly N-glycosylated mammalian proteins. We also proved that chimeric Leishmania-derived VLPs are highly immunogenic and able to elicit cross-reactive antibody response against HCV. This approach may prove useful in the development of a bivalent prophylactic vaccine against HBV and HCV and opens up a new and low-cost opportunity for the production of chimeric sHBsAg VLPs requiring N-glycosylation process for their proper functionality and immunogenicity.

Characterization of surface-exposed structural loops as insertion sites for foreign antigen delivery in calicivirus-derived VLP platform.

Chimeric virus-like particles (cVLPs) show great potential in improving public health as they are safe and effective vaccine candidates. The capsid protein of caliciviruses has been described previously as a self-assembling, highly immunogenic delivery platform. The ability to significantly induce cellular and humoral immunity can be used to boost the immune response to low immunogenic foreign antigens displayed on the surface of VLPs. Capsid proteins of caliciviruses despite sequence differences share similar architecture with structural loops that can be genetically modified to present foreign epitopes on the surface of cVLPs. Here, based on the VP1 protein of norovirus (NoV), we investigated the impact of the localization of the epitope in different structural loops of the P domain on the immunogenicity of the presented epitope. In this study, three distinct loops of NoV VP1 protein were genetically modified to present a multivalent influenza virus epitope consisting of a tandem repeat of M2/NP epitopes. cVLPs presenting influenza virus-conserved epitopes in different localizations were produced in the insect cells and used to immunize BALB/c mice. Specific reaction to influenza epitopes was compared in sera from vaccinated mice to determine whether the localization of the foreign epitope has an impact on the immunogenicity.

Effect of Selected Crosslinking and Stabilization Methods on the Properties of Porous Chitosan Composites Dedicated for Medical Applications.

Chitosan is one of the most commonly employed natural polymers for biomedical applications. However, in order to obtain stable chitosan biomaterials with appropriate strength properties, it is necessary to subject it to crosslinking or stabilization. Composites based on chitosan and bioglass were prepared using the lyophilization method. In the experimental design, six different methods were used to obtain stable, porous chitosan/bioglass biocomposite materials. This study compared the crosslinking/stabilization of chitosan/bioglass composites with ethanol, thermal dehydration, sodium tripolyphosphate, vanillin, genipin, and sodium ß-glycerophosphate. The physicochemical, mechanical, and biological properties of the obtained materials were compared. The results showed that all the selected crosslinking methods allow the production of stable, non-cytotoxic porous composites of chitosan/bioglass. The composite with genipin stood out with the best of the compared properties, taking into account biological and mechanical characteristics. The composite stabilized with ethanol is distinct in terms of its thermal properties and swelling stability, and it also promotes cell proliferation. Regarding the specific surface area, the highest value exposes the composite stabilized by the thermal dehydration method.

Bioactive glasses enriched with zinc and strontium: synthesis, characterization, cytocompatibility with osteoblasts and antibacterial properties.

Purpose: The aim of the presented work was to characterize the new obtained bioglasses and assess their biological performance in vitro. Bioglasses were produced using the sol-gel method in the SiO2-P2O5-CaO system, for the purpose as composite ingredients. Their chemical composition was enriched with ZnO to introduce antibacterial properties and SrO with osteoinductive effect. The properties of bioglasses were compared and the effect of chemical composition and particle size on their biological properties was assessed. Methods: The bioglasses were evaluated via TG-DTA, FTIR, SEM-EDS analyses before and after incubation in SBF solution. LDH and WST-1 tests were used to determine the level of cytotoxicity of the tested bioglasses on hFOB1.19 osteoblasts. Results: The results show that the developed bioglasses release Ca2+, are bioactive in SBF solution, not cytotoxic and show antibacterial activity in contact with Pseudomonas aeruginosa and Staphylococcus aureus strains. Bioglasses enriched with ZnO show the highest bactericidal activity. All tested bioglasses enhanced hFOB 1.19 cells proliferation. Particle size has a lower effect on biological performance of the bioglasses than their chemical composition. Conclusions: The conducted research showed that bioglass modification with SrO and ZnO can be considered particularly for the development of biomaterials supporting bone regeneration and the treatment of infected bone defects.

Low-volume label-free SARS-CoV-2 detection with the microcavity-based optical fiber sensor.

Accurate and fast detection of viruses is crucial for controlling outbreaks of many diseases; therefore, to date, numerous sensing systems for their detection have been studied. On top of the performance of these sensing systems, the availability of biorecognition elements specific to especially the new etiological agents is an additional fundamental challenge. Therefore, besides high sensitivity and selectivity, such advantages as the size of the sensor and possibly low volume of analyzed samples are also important, especially at the stage of evaluating the receptor-target interactions in the case of new etiological agents when typically, only tiny amounts of the receptor are available for testing. This work introduces a real-time, highly miniaturized sensing solution based on microcavity in-line Mach-Zehnder interferometer (µIMZI) induced in optical fiber for SARS-CoV-2 virus-like particles detection. The assay is designed to detect conserved regions of the SARS-CoV-2 viral particles in a sample with a volume as small as hundreds of picoliters, reaching the detection limit at the single ng per mL level.

Characterization of Immune Response towards Generation of Universal Anti-HA-Stalk Antibodies after Immunization of Broiler Hens with Triple H5N1/NA-HA-M1 VLPs.

(1) Background: Avian influenza viruses (AIVs) promptly evade preexisting immunity by constantly altering the immunodominant neutralizing antibody epitopes (antigenic drift) or by procuring new envelope serotypes (antigenic shift). As a consequence, the majority of antibodies elicited by infection or vaccination protect only against closely related strains. The immunodominance of the globular head of the main glycoprotein has been shown to mask the immunogenicity of the conserved regions located within the hemagglutinin (HA) protein. It has been shown that the broadly neutralizing universal antibodies recognize the HA2 domain in headless hemagglutinin (HA-stalk). Therefore, the HA-stalk is a highly conserved antigen, which makes it a good candidate to be used in universal vaccine development against AIVs. (2) Methods: Sf9 insect cells were used to produce triple H5N1/NA-HA-M1 influenza virus-like particles (VLPs) via co-expression of neuraminidase, hemagglutinin and matrix proteins from a tricistronic expression cassette. Purified influenza VLPs were used to immunize broiler hens. An in-depth characterization of the immune response was performed with an emphasis on the pool of elicited universal antibodies. (3) Results: Our findings suggest, that after vaccination with triple H5N1/NA-HA-M1 VLPs, hens generate a pool of broad-spectrum universal anti-HA-stalk antibodies. Furthermore, these universal antibodies are able to recognize the mammalian-derived HA-stalk recombinant proteins from homologous H5N1 and heterologous H7N9 AIVs as well as from the heterosubtypic human H1N1 influenza strain. (4) Conclusions: Our findings may suggest that highly pathogenic avian influenza H5 HA protein contain functional epitopes that are attractive targets for the generation of broad-spectrum antibodies against AIVs in their native hosts.

The Preliminary Assessment of New Biomaterials Necessitates a Comparison of Direct and Indirect Cytotoxicity Methodological Approaches.

BACKGROUND: Cytotoxicity testing is a primary method to establish the safety of biomaterials, e.g., biocomposites. Biomaterials involve a wide range of medical materials, which are usually solid materials and are used in bone regeneration, cardiology, or dermatology. Current advancements in science and technology provide several standard cytotoxicity testing methods that are sufficiently sensitive to detect various levels of cellular toxicity, i.e., from low to high. The aim was to compare the direct and indirect methodology described in the ISO guidelines UNE-EN ISO 10993-5:2009 Part 5. METHODS: Cell proliferation was measured using WST-1 assay, and cytotoxicity was measured using LDH test kit. RESULTS: The results indicate that the molecular surface of biomaterials have impact on the cytotoxicity and proliferation profile. Based on these results, we confirm that the indirect method does not provide a clear picture of the cell condition after the exposure to the surface, and moreover, cannot provide complete results about the effects of the material. CONCLUSIONS: Comparison of both methods shows that it is pivotal to investigate biomaterials at the very early stages using both indirect and direct methods to access the influence of the released toxins and surface of the material on the cell condition.

Assessment of the Toxicity of Biocompatible Materials Supporting Bone Regeneration: Impact of the Type of Assay and Used Controls.

Assessing the toxicity of new biomaterials dedicated to bone regeneration can be difficult. Many reports focus only on a single toxicity parameter, which may be insufficient for a detailed evaluation of the new material. Moreover, published data frequently do not include control cells exposed to the environment without composite or its extract. Here we present the results of two assays used in the toxicological assessment of materials' extracts (the integrity of the cellular membrane and the mitochondrial activity/proliferation), and the influence of different types of controls used on the obtained results. Results obtained in the cellular membrane integrity assay showed a lack of toxic effects of all tested extracts, and no statistical differences between them were present. Control cells, cells incubated with chitosan extract or chitosan-bioglass extract were used as a reference in proliferation calculations to highlight the impact of controls used on the result of the experiment. The use of different baseline controls caused variability between obtained proliferation results, and influenced the outcome of statistical analysis. Our findings confirm the thesis that the type of control used in an experiment can change the final results, and it may affect the toxicological assessment of biomaterial.

Performance of electrochemical immunoassays for clinical diagnostics of SARS-CoV-2 based on selective nucleocapsid N protein detection: Boron-doped diamond, gold and glassy carbon evaluation.

The 21st century has already brought us a plethora of new threats related to viruses that emerge in humans after zoonotic transmission or drastically change their geographic distribution or prevalence. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was first spotted at the end of 2019 to rapidly spread in southwest Asia and later cause a global pandemic, which paralyzes the world since then. We have designed novel immunosensors targeting conserved protein sequences of the N protein of SARS-CoV-2 based on lab-produced and purified anti-SARS-CoV-2 nucleocapsid antibodies that are densely grafted onto various surfaces (diamond/gold/glassy carbon). Titration of antibodies shows very strong reactions up to 1:72 900 dilution. Next, we showed the mechanism of interactions of our immunoassay with nucleocapsid N protein revealing molecular recognition by impedimetric measurements supported by hybrid modeling results with both density functional theory and molecular dynamics methods. Biosensors allowed for a fast (in less than 10 min) detection of SARS-CoV-2 virus with a limit of detection from 0.227 ng/ml through 0.334 ng/ml to 0.362 ng/ml for glassy carbon, boron-doped diamond, and gold surfaces, respectively. For all tested surfaces, we obtained a wide linear range of concentrations from 4.4 ng/ml to 4.4 pg/ml. Furthermore, our sensor leads to a highly specific response to SARS-CoV-2 clinical samples versus other upper respiratory tract viruses such as influenza, respiratory syncytial virus, or Epstein-Barr virus. All clinical samples were tested simultaneously on biosensors and real-time polymerase chain reactions.

Functional Severe Acute Respiratory Syndrome Coronavirus 2 Virus-Like Particles From Insect Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a major epidemic threat since the beginning of 2020. Efforts to combat the virus and the associated coronavirus disease 2019 (COVID-19) disease are being undertaken worldwide. To facilitate the research on the virus itself, a number of surrogate systems have been developed. Here, we report the efficient production of SARS-CoV-2 virus-like particles (VLPs) in insect cells. Contrary to widely used pseudovirus particles, where only one coronaviral protein is displayed within a heterologous scaffold, developed VLPs are structurally similar to the native virus and allow for more throughput studies on the biology of the infection. On the other hand, being devoid of the viral genome, VLPs are unable to replicate and thus safe to work with. Importantly, this is the first report showing that SARS-CoV-2 VLPs can be efficiently produced in insect cells and purified using scalable affinity chromatography.

Recombinant VP60 in the form of virion-like particles as a potential vaccine against rabbit hemorrhagic disease virus.

Rabbit hemorrhagic disease virus (RHDV) which causes a highly contagious disease of wild and domestic rabbits belongs to the family Caliciviridae. It is a small, positive single-stranded RNA virus with a genome of 7.5 kb and has a diameter of approximately 40 nm. In negatively stained electron micrographs the virus shows typical calicivirus morphology with regularly arranged cup-shaped structures on the surface. It is a major pathogen of rabbits in many countries. Vp60 - a coat protein of molecular mass around 60 kDa is the major antigen of RHDV. It is present as 90 dimeric units per virion particle. We have expressed VP60 gene in the baculovirus system with the aim to use it as a potential vaccine against RHDV and a diagnostic reagent in immunological tests. cDNA of the vp60 gene of strain SGM, was cloned into a baculovirus transfer vector as full-length gene, as well as truncated gene lacking 600 5'-terminal nucleotides. The sequence of SGM VP60 differed markedly from that of the reference strain. Full-length recombinant VP60 protein from the SGM strain self-assembled to form virus-like particles (VLPs). These particles observed by electron microscopy were morphologically similar to native virions and were able to agglutinate human group 0 erythrocytes. After immunization the recombinant particles induced RHDV-specific antibodies in rabbits and guinea pigs. Rabbits immunized with the VLPs were fully protected against challenge with a virulent RHDV.

Hemagglutinin stalk domain from H5N1 strain as a potentially universal antigen.

Influenza A virus infections are the major public health concern and cause significant morbidity and mortality each year worldwide. Vaccination is the main strategy of influenza epidemic prevention. However, seasonal vaccines induce strain-specific immunity and must be reformulated annually based on prediction of the strains that will circulate in the next season. Thus, it is essential to develop vaccines that would induce broad and persistent immunity to influenza viruses. Hemagglutinin is the major surface antigen of the influenza virus. Recent studies revealed the importance of HA stalk-specific antibodies in neutralization of different influenza virus strains. Therefore, it is important to design an immunogen that would focus the immune response on the HA stalk domain in order to elicit neutralizing antibodies. In the present study, we report characterization of a conserved truncated protein, potentially a universal influenza virus antigen from the H5N1 Highly Pathogenic Avian Influenza A virus strain. Our results indicate that exposure of the HA stalk domain containing conserved epitopes results in cross reactivity with different antibodies (against group 1 and 2 HAs). Additionally, we conclude that HA stalk domain contains not only conformational epitopes recognized by universal FI6 antibody, but also linear epitopes recognized by other antibodies.

A multiplex real-time PCR assay for detection of oseltamivir-resistant strains of influenza virus.

Influenza is a contagious disease of humans and animals caused by viruses belonging to the Orthomyxoviridae family. The influenza A virus genome consists of negative sense, single-stranded, segmented RNA. Influenza viruses are classified into subtypes based on two surface antigens known as hemagglutinin (H) and neuraminidase (N). The main problem with influenza A viruses infecting humans is drug resistance, which is caused by antigenic changes. A few antiviral drugs are available, but the most popular is the neuraminidase inhibitor - oseltamivir. The resistance against this drug has probably developed through antigenic drift by a point mutation in one amino acid at position 275 (H275Y). In order to prevent a possible influenza pandemic it is necessary to develop fast diagnostic tests. The aim of this project was to develop a new test for detection of influenza A virus and determination of oseltamivir resistance/sensitivity in humans. Detection and differentiation of oseltamivir resistance/sensitivity of influenza A virus was based on real-time PCR. This test contains two TaqMan probes, which work at different wavelengths. Application of techniques like multiplex real-time PCR has greatly enhanced the capability for surveillance and characterization of influenza viruses. After its potential validation, this test can be used for diagnosis before treatment.

Universal biosensor for detection of influenza virus.

Influenza is a contagious disease caught by humans caused by viruses belonging to the family Orthomyxoviridae. Each year, the influenza virus infects millions of people and kills hundreds of thousands of them. Traditional diagnostic methods, such as virus propagation and isolation, antigen capture immunoassays and molecular methods are not sufficient for the detection of the influenza virus. Development of a valid diagnostic assay for quick detection (in less than an hour) of the virus, with high sensitivity, is a challenge for researchers all over the world. Here we present a new, universal immunosensor for detection of the influenza A virus. By using electrochemical impedance spectroscopy (EIS) and direct attachment of antibodies to the gold electrode the assay allows detection of the pathogen with sensitivity similar to molecular methods in relatively short time. Application of universal anti-M1 antibodies allows detection of all serotypes of influenza A virus. The simple design of the sensor facilitates miniaturization of the device and its implementation for routine diagnostics during first contact with the patient, before applying a proper treatment.

Expression of avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) under control of the ptcB promoter in Lactococcus lactis.

Gram-positive and nonpathogenic lactic acid bacteria (LAB) are considered to be promising candidates for the development of new, safe systems of heterologous protein expression. Recombinant LAB has been shown to induce specific local and systemic immune response against selected pathogens, and could be a good alternative to classical attenuated carriers. The main goal of our study was to express the avian influenza haemagglutinin (H5) and chicken interleukin 2 (chIL-2) in Lactococcus lactis. Results of this study were anticipated to lead to construction of lactococcal strain(s) with potential vaccine properties against the avian influenza A (H5N1) virus. Expression of the cloned H5 gene, its His-tagged variant and chIL-2 gene, under the control of the ptcB gene promoter was attested by RT-PCR on transcriptional level and Western or dot blot analysis on translational level, demonstrating that system can be an attractive solution for production of heterologous proteins. The results of the preliminary animal trial conducted in mice are a promising step toward development of a vaccine against avian bird flu using Lactococcus lactis cells as antigen carriers.

Anti-influenza A virus activity of uridine derivatives of 2-deoxy sugars.

Influenza viruses are important pathogens that cause respiratory infections in humans and animals. Apart from vaccinations, antiviral drugs play a significant role in controlling spread of the disease. Influenza A virus contains two membrane glycoproteins on the external part of viral envelope: hemagglutinin (HA) and neuraminidase (NA), which are crucial for productive infection in target cells. In the present work, two derivatives of tunicamycin - uridine derivatives of 2-deoxy sugars (designated IW3 and IW7), which target the glycan processing steps during maturation of viral glycoproteins, were assayed for their ability to inhibit influenza A virus infection in vitro. Using the cytopathic effect (CPE) inhibition assay and viral plaque reduction assay we showed, that both IW3 and IW7 inhibitors exerted significant inhibitory effect on influenza A virus infection in MDCK cells without significant toxicity for the cells. Moreover, tested compounds selectively suppressed viral protein expression in a dose-dependent manner, suggesting that the mechanism of their antiviral activity may be similar to this shown previously for other viruses. We have also excluded the possibility that both inhibitors act at the replication step of virus life cycle. Using real-time PCR assay it was shown that IW3 and IW7 did not change the level of viral RNA in infected MDCK cells after a single round of infection. Therefore, inhibition of influenza A virus infection by uridine derivatives of 2-deoxy sugars, acting as glycosylation inhibitors, is a promising alternative approach for the development of new anti-influenza A therapy.

Detection and differentiation of virulent and avirulent strains of Newcastle disease virus by real-time PCR.

A rapid diagnostic method based on the melting curve SYBR Green I real-time PCR analysis was developed to detect and differentiate Newcastle disease virus (NDV) strains. Degenerated primers based on the cleavage site sequence of the F0 gene were designed to detect specific sequences characteristic of virulent and avirulent strains of NDV. Eighteen strains of NDV from four lineages were identified and grouped into virulent and avirulent strains. Peaks on the melting temperature graph with melting temperature values between 80.00 and 83.80°C were observed for lentogenic (avirulent) strains. T(m) values higher than 83.80 were observed for virulent (mesogenic and velogenic) strains. The detection limit of real-time PCR was 2 × 10(2) plasmid copies per reaction or 10(2) EID(50) for velogenic strains and 10(3) EID(50) for lentogenic strains. The results obtained in this study demonstrate the possible applications for melting curve real-time PCR analysis in laboratory practice for the diagnosis and differentiation of avirulent and virulent strains of Newcastle disease virus.