Littérature scientifique sur le sujet « P18 viral capsid antigen »

Using recombinant 15- to 30-kDa fragments and fusion with glutathione S-transferase (GST), we investigated the seroreactivity of three large structural proteins of Epstein-Barr virus (EBV), p150 (BcLF1, capsid), p143 (BNRF1, tegument), and gp125 (BALF4, membrane) in Western blots. None of 13 fragments tested, however, was qualified for diagnostic application. In contrast, the two small viral capsid antigens (VCA), p18 (BFRF3) and p23 (BLRF2), demonstrated sensitive (100%) EBV-specific immunoglobulin G (IgG) reactivities. While p18 additionally showed maximum sensitivity for IgM detection, the IgM sensitivity of p23 was restricted (44%). An autologous fusion protein, p23-p18, which consists N-terminally of full-length p23, followed by the carboxy half of p18, was constructed. This antigen was subjected to indirect VCA enzyme-linked immunosorbent assays (ELISAs), for IgG and IgM, as well as to a μ-capture (μc) IgM ELISA. All assays were found to be 100% specific when EBV-negative sera were tested. Using sera from previously infected individuals, the p23-p18 fusion revealed an improved IgG sensitivity of 99% compared to sensitivities of 97 and 93% for the single antigens p18 and p23, respectively. The sensitivity and specificity of the indirect IgM ELISA with samples of primary and past infections, respectively, were 100%. The μc principle for IgM overcame completely the interference by rheumatoid factors. Compared to the specificity of the indirect IgM version, the specificity with sera collected from rheumatoid arthritis patients increased from 48 to 100%. In summary, the p23-p18 IgG and μc IgM ELISAs showed excellent performances and are promising new diagnostic tests for the detection of EBV-specific antiviral capsid antibodies.

18186 Background: Pulmonary lymphoepithelioma-like carcinoma (PLELC) belongs to large cell sub-type of non-small cell lung cancer (NSCLC) with just about 160 cases reported previously. Its epidemiology, prognosis and therapeutic approach is evidently different from other NSCLCs. EBV infection is seen in 100% of Asian PLELC patients whenever tested. The aim of this study was to analyze serum antibodies level against EBV in PLELC patients. Methods: Sera of all seven patients with re-confirmed PLELC from a single institute in Canton, China in the past 4 years were collected. Serum levels of 8 different antibodies against EBV (Epstein-Barr virus): EBNA1 (EBV nuclear antigen 1- IgA and IgG), Zta (Bam Z transactivator antigen-IgA and IgG), VCA-p18 (Viral capsid antigen-p18-IgA and IgG), VCA-antigen complex (VCA-IgA) and Early antigen complex (EA-IgA ) were measured in each of these patients using enzyme-linked immunosorbent assay and Immunoenzymatic assay respectively. Results: Out of 7 cases, 6 showed high EBNA-1 level, 4 showed high zta, 3 showed high VCA-p18, 3 showed high titer of VCA complex and 2 showed high EA complex titer ( Table 1 ) Conclusions: Though small sample size, this is the first study in this depth ever to conclude that sera of all Asian patients with pulmonary LELC have high antibody titers against EBV. The infection mainly is latency II type as seen in Nasopharyngeal carcinoma and Hodgkin’s disease as well. A small portion of PLELC express lytic products such as Zta, EA and VCA. The authors assume that pre-therapeutic measurement of serum levels of anti-EBV antibodies (at least 2 kinds) is highly warranted in suspected Asian NSCLC patients. Positive result strongly puts PLELC into differential diagnosis. [Table: see text] No significant financial relationships to disclose.

ABSTRACTOver 35% of children in a region of malaria endemicity are infected with Epstein-Barr virus (EBV) by 6 months of age. This susceptibility may be linked to impaired transplacental transfer of antibodies. In this study, we determined the effect of malaria exposure during pregnancy on the transfer of EBV-specific maternal antibodies in a region of western Kenya that experiences endemic malaria. Pregnant mothers were recruited and followed up until delivery to determine levels of neonatal malaria exposure. Levels of EBV lytic (viral capsid antigen [VCA], Z transcriptional activator [Zta], and early diffuse antigen complex [EAd]) and EBV latent (EBV nuclear antigen-1 (EBNA1]) and tetanus-specific IgG antibodies were measured in 70 paired maternal and cord blood samples using a Luminex-bead-based assay. A high proportion (63%) of the infants were exposed to malariain utero. Levels of EBV- and tetanus-specific antibodies were similar in malaria-infected mothers and in mothers who had no detectable malaria infection. Malaria-exposed neonates had significantly lower levels of anti-EBNA1, anti-Zta, and anti-EAd antibodies than were seen in their mothers.In uteromalaria exposure resulted in significant reductions in transplacental transfer of anti-VCA-p18 and anti-EBNA1 antibodies of 13% and 22%, respectively. Neonates received significantly low levels of anti-Zta and anti-EAd antibodies irrespective of malaria exposure levels. In multivariate analysis,in uteromalaria exposure was associated with a significant reduction in the transfer of anti-VCA-p18 and anti-EBNA1 antibodies to the neonates (P= 0.0234 andP= 0.0017, respectively). Malaria during pregnancy results in differential levels of transfer of EBV-specific antibodies from the mother to the fetus. The impaired transplacental transfer of some antibodies may lead to the malaria-exposed neonates being susceptible to early EBV infection.

ABSTRACT Undifferentiated nasopharyngeal carcinoma (NPC; WHO type III) is 100% associated with Epstein-Barr virus (EBV) infection and the fourth most prevalent cancer in Indonesian males. Therapy failure is high, since most patients come to the hospital at an advanced stage of disease. Screening for early-stage NPC is needed. Here, a simple and economical two-step enzyme-linked immunosorbent assay (ELISA) system is proposed for diagnosing NPC in high-risk populations, employing the peptide-based immunoglobulin A (IgA) EBNA1 plus viral capsid antigen p18 ELISA as an initial screening test and the IgA early antigen (EA) ELISA using a different set of EBV antigens as a confirmation test. A total of 151 NPC patients and 199 regional healthy EBV carriers were used to evaluate the two-step ELISA approach. Routinely, EBV IgG immunoblotting is used as a standard confirmation test. The sensitivity and specificity for diagnosing NPC by the two-step ELISA approach increased from 85.4% to 96.7% and 90.1% to 98%, respectively, with positive predictive values and negative predictive values increasing from 78.7 and 93.9% to 97.3 and 97.5%, respectively, relative to the immunoblotting confirmation system. On discrepant samples, additional testing was done by EBV DNA load quantification in blood. Results showed that 5/11 discrepant NPC samples with an elevated IgA EA ELISA also had elevated an EBV DNA load in the circulation (range, 3,200 to 25,820 copies/ml). Therefore, the IgA EA ELISA is proposed as a confirmation test in first-line NPC serological screening studies. This two-step EBV ELISA system provides a standardized approach for NPC screening and may be used in combination with dried blood sampling in future field studies for identification of early-stage NPC in high-risk regions.

Background The simultaneous infection of Plasmodium falciparum and Epstein-Barr virus (EBV) could promote the development of the aggressive endemic Burkitt’s Lymphoma (eBL) in children living in P. falciparum holoendemic areas. While it is well-established that eBL is not related to other human malaria parasites, the impact of EBV infection on the generation of human malaria immunity remains largely unexplored. Considering that this highly prevalent herpesvirus establishes a lifelong persistent infection on B-cells with possible influence on malaria immunity, we hypothesized that EBV co-infection could have impact on the naturally acquired antibody responses to P. vivax, the most widespread human malaria parasite. Methodology/Principal findings The study design involved three cross-sectional surveys at six-month intervals (baseline, 6 and 12 months) among long-term P. vivax exposed individuals living in the Amazon rainforest. The approach focused on a group of malaria-exposed individuals whose EBV-DNA (amplification of balf-5 gene) was persistently detected in the peripheral blood (PersVDNA, n = 27), and an age-matched malaria-exposed group whose EBV-DNA could never be detected during the follow-up (NegVDNA, n = 29). During the follow-up period, the serological detection of EBV antibodies to lytic/ latent viral antigens showed that IgG antibodies to viral capsid antigen (VCA-p18) were significantly different between groups (PersVDNA > NegVDNA). A panel of blood-stage P. vivax antigens covering a wide range of immunogenicity confirmed that in general PersVDNA group showed low levels of antibodies as compared with NegVDNA. Interestingly, more significant differences were observed to a novel DBPII immunogen, named DEKnull-2, which has been associated with long-term neutralizing antibody response. Differences between groups were less pronounced with blood-stage antigens (such as MSP1-19) whose levels can fluctuate according to malaria transmission. Conclusions/Significance In a proof-of-concept study we provide evidence that a persistent detection of EBV-DNA in peripheral blood of adults in a P. vivax semi-immune population may impact the long-term immune response to major malaria vaccine candidates.

Background: Nasopharyngeal carcinoma (NPC), especially the WHO type III, is correlated almost100% with Epstein Barr Virus (EBV) infection. This is indicated by high IgG and IgA antibody responsesagainst viral capsid antigen (VCA), early antigen (EA) and Epstein Barr Nuclear antigen (EBNA).Increased IgA NPC character antibodies may be detected 2-10 years before the presence of the tumor. Thisoccurs as a result of reactivation of EBV infection. Purpose: To find out the level of IgA NPC characterantibodies (EBNA1+VCA p-18) in patients with chronic symptoms in the head and neck and to determine whether the level of IgA can be used as an early sign of NPC. Methods: Observational analytic study on 218 patients with chronic symptoms in the head and neck. The research was conducted from July 2006to September 2010. ELISA technique was used as serology test for IgA (EBNA1+VCA p-18). Result: Samples were 90 males and 128 females. High level of IgA by ELISA was found in 28 males (31.1%) and 45 females (35.2%). The IgA level tended to increase with age. The most common chronic symptoms inthe head and neck were chronic rhinitis (15.6%) and nasal obstruction (7.8%). From all patients who hadhigh level of IgA, 3 patients (4.1%) were found positive of early stage NPC. Conclusion: More than 33%of patients with chronic symptoms of head and neck had high level of IgA NPC character. This methodcan be used as an early detection of NPC. Keywords: serology test in NPC, EBNA1, VCA p-18, NPC symptoms in head and neck Abstrak : Latar belakang: Karsinoma nasofaring (KNF) terutama tipe WHO III berkorelasi hampir 100%dengan infeksi Epstein Barr Virus (EBV). Hal ini ditunjukkan dengan tingginya respons antibodi IgGdan IgA terhadap viral capsid antigen (VCA), early antigen (EA) EBV serta antibodi Epstein BarrNuclear Antigen (EBNA). Kenaikan antibodi IgA dengan karakter KNF dapat terjadi 2-10 tahun sebelumterjadinya tumor. Hal ini terjadi sebagai akibat adanya reaktivasi infeksi EBV. Tujuan: Mengetahui kadarIgA karakter KNF (EBNA1+VCA p-18) pada penderita dengan gejala kronis di daerah kepala dan leherdan mengetahui apakah kadar IgA dapat digunakan sebagai tanda awal terjadinya KNF. Metode: Suatukajian analitik observasional terhadap 218 penderita dengan gejala kronis di daerah kepala dan leher.Penelitian ini dilakukan Juli 2006 sampai dengan September 2010. Pemeriksaan serologi IgA (EBNA1+VCA-p18)dilakukan denganteknik ELISA.Hasil:Terdapat90penderita laki-lakidan128 penderitaperempuan.HasiltesserologiIgAELISAdengankadartinggiditemukanpada28laki-laki(31,1%)dan45perempuan (35,2%). Kadar IgA cenderung meningkat pada peningkatan usia. Gejala kronis yangterbanyak dikeluhkan penderita adalah rinitis kronis, yaitu sebanyak 34 penderita (15,6%), diikuti denganobstruksi hidung sebanyak 17 penderita (7,8%). Pemeriksaan klinis lebih lanjut dari penderita yangmempunyai kadar IgA tinggi menunjukkan bahwa 3 penderita (4,1%) positif terkena kanker nasofaringstadium awal. __ Lebih dari 33% penderita dengan gejala kronis di daerah kepala dan lehermemiliki kadar IgA karakter KNF yang tinggi. Kadar IgA karakter KNF yang tinggi dapat digunakansebagai penanda awal kejadian KNF. Kata kunci: uji serologi KNF,EBNA1, VCA p-18, gejala KNF

Introduction: The purpose of this study is to evaluate the diagnostic value of macrophage migration inhibitory factor in patients with nasopharyngeal carcinoma. Materials and Methods: The expression levels of macrophage migration inhibitory factor in nasopharyngeal carcinoma cell lines, tumor tissues, and plasma were measured by real-time polymerase chain reaction, Western blotting, enzyme-linked immunosorbent assay, and immunohistochemistry. Plasma Epstein-Barr virus viral capsid antigen was determined by immunoenzymatic techniques. Results: Both the messenger RNA and protein expression levels of macrophage migration inhibitory factor were upregulated in nasopharyngeal carcinoma cell lines and nasopharyngeal carcinoma tissues. Macrophage migration inhibitory factor in plasma was significantly elevated in patients with nasopharyngeal carcinoma compared to Epstein-Barr virus viral capsid antigen–negative and Epstein-Barr virus viral capsid antigen–positive healthy donors. The combination of macrophage migration inhibitory factor and Epstein-Barr virus viral capsid antigen was better for diagnosing nasopharyngeal carcinoma (area under receiver operating characteristic curve = 0.925, 95% CI: 0.898-0.951) than macrophage migration inhibitory factor (area under receiver operating characteristic curve = 0.778, 95% CI: 0.732-0.824) and Epstein-Barr virus viral capsid antigen. Combining macrophage migration inhibitory factor and Epstein-Barr virus viral capsid antigen had higher specificity (82.40% vs 69.96%) and higher positive predictive value (79.17% vs 67.44%) without an obvious reduction in sensitivity (95.25%) compared to Epstein-Barr virus viral capsid antigen alone. Macrophage migration inhibitory factor was highly expressed in nasopharyngeal carcinoma cell lines, whereas it was not associated with Epstein-Barr virus infection. The level of macrophage migration inhibitory factor in plasma was not related to the titer of Epstein-Barr virus viral capsid antigen. Conclusion: The combination of macrophage migration inhibitory factor and Epstein-Barr virus viral capsid antigen increases the specificity and positive predictive value of detecting nasopharyngeal carcinoma and improves the diagnostic accuracy of nasopharyngeal carcinoma in high-risk individuals.

ABSTRACT The Gag polyprotein of murine leukemia virus (MLV) is processed into matrix (MA), p12, capsid (CA), and nucleocapsid (NC) proteins. p12 affects early events of virus replication and contains a PPPY motif important for virus release. To probe the functions of p12 in the early steps of MLV replication, we tested whether p12 can be replaced by spleen necrosis virus (SNV) p18, human immunodeficiency virus type 1 p6, or Rous sarcoma virus p2b. Analyses revealed that all chimeras generated virions at levels similar to that of MLV gag-pol; however, none of them could support MLV vector replication, and all of them exhibited severely reduced DNA synthesis upon virus infection. Because a previously reported SNV gag-MLV pol chimera, but not the MLV hybrid with SNV p18, can support replication of an MLV vector, we hypothesized that other Gag proteins act cooperatively with p12 during the early phase of virus replication. To test this hypothesis, we generated three more MLV-based chimeras containing SNV CA, p18-CA, or p18-CA-NC. We found that the MLV chimera containing SNV p18-CA or p18-CA-NC could support MLV vector replication, but the chimera containing SNV CA could not. Furthermore, viruses derived from the MLV chimera with SNV CA could synthesize viral DNA upon infection but were blocked at a post-reverse-transcription step and generated very little two long terminal repeat circle DNA, thereby producing a phenotype similar to that of the provirus formation-defective p12 mutants. Taken together, our data indicate that when p12/p18 or CA was from different viruses, despite abundant virus production and proper Gag processing, the resulting viruses were not infectious. However, when p12/p18 and CA were from the same virus, even though they were from SNV and not MLV, the resulting viruses were infectious. Therefore, these results suggest a cooperative effect of p12 and CA during the early events of MLV replication.

Antigene e anticorpo sono i due reagenti chiave di un saggio immunodiagnostico. L’indagine di nuove tecniche e il miglioramento di processi quali la purificazione e la marcatura sito-specifica di antigeni e anticorpi possono promuovere lo sviluppo di nuovi reagenti più efficienti capaci di migliorare la performance dei saggi immunodiagnostici. La prima parte di questa tesi è stata focalizzata sull’esplorazione di tecniche biotecnologiche innovative nella produzione di antigeni al fine di migliorare i saggi per la rilevazione degli anticorpi IgM e IgG specifici per il virus Epstein-Barr. Il virus EBV è causa della mononucleosi infettiva ed è associato ad un crescente numero di tumori; per questa motivo è importante sviluppare saggi diagnostici per la rilevazione di EBV ad alta specificità e sensibilità. La proteina VCA p18 è uno degli antigeni più importanti per la diagnosi di EBV. I saggi attuali Diasorin LIAISON EBV VCA IgM and IgG si basano su un singolo antigene corrispondente alla regione C-terminale immunodominante della proteina p18, immobilizzata su fase solida. I vari metodi esplorati in questa tesi hanno permesso di ottenere diverse varianti dell’antigene p18 con lo scopo di migliorare le prestazioni dei saggi EBV VCA IgM e IgG a diversi livelli: 1_produzione dell’antigene p18; 2_immobilizzazione dell’antigene p18 su fase solida; 3_formato di saggio. 1_La lunghezza della regione C-terminale della proteina p18 (57aa), risulta essere considerevole per il processo sintetico ma, allo stesso tempo, troppo piccola per essere prodotta in modo efficiente per via ricombinante. Per superare questo problema, abbiamo esplorato il sistema Elastin Like Polypeptides (ELP)-Inteina basato sull’uso di una proteina capace di effettuare auto-cleavage (inteina) e un tag responsivo alla temperatura (ELP). Questa tecnica si è rivelata un eccellente sistema per la produzione del peptide p18. 2_Lo sviluppo di diverse varianti dell’antigene p18 ha permesso di esplorare varie tecniche per l’immobilizzazione dello stesso antigene su fase solida: coating covalente diretto, attraverso il complesso streptavidina-biotina e attraverso l’uso dei peptidi leucine zipper (o velcro). L’immobilizzazione del peptide p18 su fase solida attraverso questi vari metodi è avvenuta con successo e l’attività immunochimica dell’antigene, immobilizzato con queste tecniche innovative, è comparabile o migliore rispetto a quella del peptide sintetico utilizzato nei saggi attuali. 3_Nonostante il saggio Diasorin LIAISON EBV VCA IgM abbia una buona performance analitica, al fine di ottenere un aumento di specificità, è stato esplorato un nuovo tipo di formato di saggio. Sfortunatamente i risultati indicano che questo diverso tipo di formato raggiunge un livello di specificità minore rispetto a quello del saggio attuale. La seconda parte di questa tesi è stata focalizzata sull’esplorazione di un metodo innovativo per la marcatura sito specifica degli anticorpi. Uno degli approcci più promettenti è basato sulla generazione di gruppi tiolo liberi attraverso la riduzione parziale e selettiva dei ponti disolfuro inter-catena presenti a livello della “hinge region” e la loro reazione con molecole marcanti caratterizzate dal possedere gruppi funzionali reattivi verso i gruppi sulfidrilici. Questa tecnologia è stata utilizzata per la biotinilazione di due diversi anticorpi usati attualmente per il rilevamento della proteina virale p24 di HIV e per l’antigene FGF23. I risultati suggeriscono che la biotinilazione sito-specifica rispetto a quella classica random promuove un miglioramento dell'attività immunochimica degli anticorpi con una conseguente ottimizzazione della performance dei saggi immunodiagnostici.
Antigen and antibody are the two key reagents for an immunodiagnostic assay. Investigation of new techniques and improvement of processes such as purification and site-specific labeling of antigen and antibody molecules can promote the development of new more powerful bioreagents able of improving the performance of immunodiagnostic assays. The first part of this thesis aimed to explore innovative biotechnology techniques in antigen production for the improvement of immunoassays that allow the detection of antibodies directed against the Epstein-Barr virus. EBV virus is the causative agent of infectious mononucleosis and it is considered to be associated with a still increasing number of tumors; for this reason it is important to develop diagnostic assays for EBV detection with high specificity and sensitivity. The viral capsid protein VCA p18 is one of the most important antigens for the diagnosis of EBV. The current Diasorin LIAISON EBV VCA IgM and IgG assays rely on a single antigen, consisting in a synthetic peptide corresponding to the immunodominant C-terminal portion of the p18 protein, which is immobilized on solid phase (indirect format). The several methods explored in this thesis have allowed to obtain different variants of the p18 antigen with the aim to improve the performance of DiaSorin LIAISON EBV VCA IgM and IgG assays at different levels: 1_production of p18 antigen; 2_immobilization of p18 antigen on solid phase; 3_immunoassay format. 1_ The length of the immunodominant C-terminal portion of the p18 protein (57aa) appears to be considerable for the synthetic route but, at the same time, too small to be effectively produced in a recombinant fashion. To overcome this problem, we explored the Elastin Like Polypeptides (ELP)-Intein system, a method based on the use of a self-cleavable protein (the intein) and a temperature responsive tag (ELP). This technique has proved to be an excellent system for the preparation of the p18 peptide. 2_The development of different variants of p18 antigen has enabled to explore different techniques for the immobilization of the same antigen on solid phase: direct covalent coating, through streptavidin-biotin complex and through an innovative procedure based on the use of leucine zipper (or “velcro”) peptides. The immobilization of the p18 peptide on solid phase through these different methods occurred successfully and the immunochemical activity of the antigen, immobilized with these innovative techniques, is comparable or better than that of the synthetic peptide used in the current immunoassays. 3_Despite the DiaSorin LIAISON EBV VCA IgM immonoassay has a good analytical performance, in order to obtain an increase of specificity, a new assay format was explored. Unfortunately, the results indicate that this different type of format reaches a lower level of specificity than that of current assay. The second part of this thesis aimed to explore an innovative method for the site-specific labeling of antibodies. One the most promising approach is based on the generation of free thiol groups by selective partial reduction of the interchain disulfide bridges present at the level of the “hinge region” and their reaction to labels carrying sulfhydryl-reactive chemical groups. This technology was used for the biotinylation of two different antibodies currently used in immunoassays for the HIV p24 viral protein and for FGF23 antigen detection. The results suggest that the site-specific biotinylation compared to the random traditional biotinylation promotes a great improvement of antibodies immunochemical activity with a consequent optimization of immunodiagnostic assays performance.

The Picornaviridae is a family of viruses of economic importance that have a major impact on human and animal health. Some of the major genera found in the Picornaviridae family are Enterovirus which includes Poliovirus (PV) and Human Rhinovirus (HRV), Cardiovirus which includes Theiler’s murine encephalomyelitis virus (TMEV) and Saffold virus (SAFV), Aphthovirus of which the Foot and Mouth disease virus (FMDV) is a member and Hepatovirus which includes Hepatitis A virus (HAV). Picornaviruses have a single stranded, positive sense RNA genome which is approximately 7.5-8.4 kb pairs in size. The picornavirus genome is translated into a large polyprotein and is proteolytically cleaved by viral proteases namely 2Apro, 3Cpro and 3CDpro into mature viral structural and non-structural polypeptides encoded by the P1, P2 and P3 domains. Picornaviruses utilise host cell machinery and cellular pathways for entry and uncoating, genome replication and capsid assembly. In our laboratory, we are studying the mechanisms by which TMEV interacts with host cell components and our recent research shows that molecular chaperones are required for a production infection. To follow up on this observation, the overall aim of this study was to prepare antigen for the generation of polyclonal antibodies against the TMEV VP1 and 3Cpro proteins. To this end, the TMEV VP1 and 3Cpro amino acid sequences were analysed to identify hydrophobic, hydrophilic and antigenic regions. Homology modelling was performed in order to predict linear B cell epitopes exposed on the surface of the protein structures. The full length coding sequences of VP1 and 3Cpro were selected for amplification by the PCR and cloning into pQE-80L for expression in a bacterial system. Time course induction studies of recombinant VP1 and 3Cpro showed that the proteins were maximally expressed at 6 hrs and 4 hrs respectively. Recombinant VP1 was solubilised using the detergent, Sarcosyl and purified by Nickel affinity chromatography under native conditions. Because recombinant VP1 co-purified with an unidentified protein, the pET expression system was used. Although no protein of the estimated size was observed by SDS-PAGE analysis in the time course induction study, Western analysis using anti-His6 (2) antibodies detected a signal of ~35 kDa. Solubility studies resulted in the presence of two protein bands in the insoluble fraction resolved between 35 and 40 kDa. Recombinant 3Cpro expressed in a bacterial system was predominantly present in the insoluble fraction. Treatment with Sarcosyl had no effect on the solubility of the recombinant protein and it was therefore purified under denaturing conditions using 8M urea. Following dialysis, 3Cpro was used for immunisation of rabbits. Crude anti-TMEV 3Cpro antibodies were able to detect as little as 107 ng of bacterially expressed antigen at a dilution of 1:100 000 by Western analysis. The presence of contaminating proteins was reduced using pre-cleared anti-TMEV 3Cpro antibodies. The antibodies were unable to detect virally expressed 3Cpro in BHK-21 cell lysate supernatant. In an attempt to determine whether TMEV 3Cpro is present in the insoluble fraction, anti-TMEV 3Cpro antibodies were tested using total protein prepared from infected and mock-infected cell lysates. Once again, no protein band the size of 3Cpro was detected. The antibodies were further tested for detection of 3Cpro in TMEV-infected cells by indirect immunofluorescence and confocal microscopy. A diffuse cytoplasmic and perinuclear distribution, as well as nuclear staining, was observed in infected BHK-21 cells. This staining pattern resembled that observed for the HRV, FMDV and EMCV 3Cpro in similar experiments. Further experiments are required to confirm specificity of these antibodies for virally-expressed 3Cpro by Western analysis and indirect immunofluorescence.