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E Brocchi

Publications and source records attributed to E Brocchi.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification of neutralizing epitopes on a European strain of swine vesicular disease virus.

Six neutralizing monoclonal antibodies (MAbs) were used to isolate MAb neutralization-resistant (MAR) mutants from a recent European strain of swine vesicular disease virus (SVDV), ITL/9/93. Sequencing of MAR mutants identified two epitopes located at positions analogous to sites 2A (VP2) and 3B (VP3) on poliovirus (PV) which have been previously identified on a Japanese strain of SVDV. A third epitope near to the C terminus of VP1, not previously recognized on SVDV, was tentatively identified in a region analogous to site 1 of PV. A fourth epitope, located in the C-terminal region of VP3, has never before been recognized as a site of neutralization on picornaviruses. All four epitopes were predicted to be surface-exposed.

Animals↗
PubMed1998

Molecular analysis of encephalomyocarditis viruses isolated from pigs and rodents in Italy.

Partial nucleotide sequences of encephalomyocarditis (EMC) viruses isolated from five, apparently independent, outbreaks of fatal myocarditis in pigs in Italy were compared with three EMC viruses isolated from wild rodents from a different geographic region in the same country. These viruses were also compared with EMC viruses isolated from pigs in other European countries and three historical strains. All the Italian EMC viruses were closely related (> 94.6% nucleotide identity), but were distinct from viruses occurring in Belgium in 1991 (< 80.5% nucleotide identity), Greece in 1990 (< 83.3% nucleotide identity) and the three older viruses (< 82.9% nucleotide identity). An EMC virus isolated from pigs in the Netherlands in 1988, was closely related to the Italian viruses (95.3-99.3% nucleotide identity). It is suggested that pigs may play a role in the movement of EMC viruses between different geographic regions.

Animals↗
PubMed1998

A further step in the evolution of rabbit hemorrhagic disease virus: the appearance of the first consistent antigenic variant.

Rabbit hemorrhagic disease virus (RHDV) is a noncultivable calicivirus that infects rabbits (Oryctolagus cuniculus) and causes epidemics of an acute fatal hepatitis. In 1997 we identified two RHDV isolates from geographically distant Italian regions, which differed antigenically from the reference strain RHDV.Bs89. In fact, they were not reactive with mAb 1H8, that is able to protect rabbits from RHD and showed a low reactivity with the rabbit convalescent serum raised against RHDV.Bs89. Experimental infection of rabbits with either RHDV isolates confirmed their high pathogenicity and their peculiar antigenic profile; nevertheless, rabbits vaccinated with the current vaccine were protected against challenge infection with these isolates. Sequence comparison definitely demonstrated that the two isolates originated from the same RHDV variant and that the similarity of their structural protein (VP60) sequences with the RHDV.Bs89 is equal to 93%. This variant was named RHDVa since shows consistent genetic and antigenic differences from the wild-type RHDV. In particular, 44% of amino acid substitutions in RHDVa VP60 were located between amino acids 344 and 370, where the similarity with RHDV.Bs89 drops to 70%, suggesting that this region probably contains the epitope recognized by mAb 1H8. In addition, this paper presents preliminary data concerning the amino acids of VP60 involved in the hemagglutination site of the virus.

Amino Acid Sequence↗
PubMed1998

A similar pattern of interaction for different antibodies with a major antigenic site of foot-and-mouth disease virus: implications for intratypic antigenic variation.

The three-dimensional structures of the Fab fragment of a neutralizing antibody raised against a foot-and-mouth disease virus (FMDV) of serotype C1, alone and complexed to an antigenic peptide representing the major antigenic site A (G-H loop of VP1), have been determined. As previously seen in a complex of the same antigen with another antibody which recognizes a different epitope within antigenic site A, the receptor recognition motif Arg-Gly-Asp and some residues from an adjacent helix participate directly in the interaction with the complementarity-determining regions of the antibody. Remarkably, the structures of the two antibodies become more similar upon binding the peptide, and both undergo considerable induced fit to accommodate the peptide with a similar array of interactions. Furthermore, the pattern of reactivities of five additional antibodies with versions of the antigenic peptide bearing amino acid replacements suggests a similar pattern of interaction of antibodies raised against widely different antigens of serotype C. The results reinforce the occurrence of a defined antigenic structure at this mobile, exposed antigenic site and imply that intratypic antigenic variation of FMDV of serotype C is due to subtle structural differences that affect antibody recognition while preserving a functional structure for the receptor binding site.

Amino Acid Sequence↗
PubMed1998

Diagnostic potential of Mab-based ELISAs for antibodies to non-structural proteins of foot-and-mouth disease virus to differentiate infection from vaccination.

This paper summarises the development of monoclonal antibody (Mab)-based immunoassays measuring antibodies to non-structural proteins of FMDV to differentiate infection from vaccination. Of the three non-structural proteins 2C, 3C and 3ABC evaluated in this study, the polypeptide 3ABC was the most immunogenic. Three ELISAs for the detection of antibodies to 3ABC were developed. Two assays rely on the competition of test sera against either a anti-3A Mab or against antisera to 3ABC raised in rabbits and guinea-pigs. The third, 3ABC Mat-ELISA, based on the direct binding of antibodies to the 3ABC trapped by a specific Mab, provided the best combination of specificity and sensitivity. The 3ABC Mat-ELISA was extensively validated for cattle, either in experimental and in field conditions, showing specificity of 99% in vaccinated and in naive cattle and the capacity to detect silent infections in FMD-vaccinated populations. The test showed similar specificity and sensitivity in experimentally vaccinated and infected sheep.

Animals↗
PubMed1997

The non-structural polyprotein 3ABC of foot-and-mouth disease virus as a diagnostic antigen in ELISA to differentiate infected from vaccinated cattle.

A diagnostic assay to differentiate antibodies induced by foot-and-mouth disease virus (FMDV) infection from those induced by vaccination was developed. The test is an indirect-trapping ELISA which uses a monoclonal antibody to trap the non-structural 3ABC-FMDV polypeptide expressed in E. coli. Experimental and field sera from naive, vaccinated and infected cattle were examined. Using the established threshold of 0.20 optical density units, the sensitivity of the assay was 100%, as all the experimental post-infection sera (n degree = 137) gave values greater than this threshold, irrespective of the FMDV serotype used for the infection. In contrast, more than 99% of sera from vaccinated animals were negative (225 out of 228 primo-vaccinates and 159 out of 159 multi-vaccinates). A high degree of specificity was also confirmed by the finding that 99.5% (442 out of 444) of sera from naive animals gave negative results. Serum conversion against 3ABC was first detected 8 days post-infection and demonstrable levels of 3ABC specific antibodies were detectable at least 1 year post-infection. The described 3ABC-ELISA is safe, cheap and also easy to perform in large scale serological surveys. The high specificity and sensitivity makes this test an ideal tool for FMD eradication campaigns and control programs.

Animals↗
PubMed1997

Molecular epidemiology of recent outbreaks of swine vesicular disease: two genetically and antigenically distinct variants in Europe, 1987-94.

Viruses from the recent epidemic of swine vesicular disease (SVD) in Europe have been isolated and characterized by antigenic and genetic methods to examine the likely epidemiological origins of the disease. Antigenic analysis was performed on 77 SVD viruses (SVDV) isolated in Europe between 1966 and 1994 using two panels of monoclonal antibodies (MAb) in a trapping ELISA. Genetic analysis of 33 of the SVD viruses by reverse transcription-polymerase chain-reaction (RT-PCR) amplification and nucleotide sequencing of the ID (VP1) coding region was also performed. Comparison of the nucleotide sequences with each other and with three other previously published SVDV sequences revealed four distinct groups which correlated exactly with the results of the pattern of reactivity with MAbs. The first group consisted solely of the earliest SVD virus isolated (ITL/1/66) while the second group comprised viruses present in Europe and Japan between 1972 and 1981. The third group consisted of viruses isolated from outbreaks of SVD in Italy between December 1988 and June 1992. Viruses isolated between 1987 and 1994 from Romania, the Netherlands, Italy and Spain formed a fourth group. The genetic and antigenic similarity of the most recent virus isolates from Western Europe to a virus isolated in Romania 5 years previously suggests that the possible origin of the recent epidemic of swine vesicular disease in Western Europe was in Eastern Europe.

Animals↗
PubMed1995

Development of two novel monoclonal antibody-based ELISAs for the detection of antibodies and the identification of swine isotypes against swine vesicular disease virus.

Two novel formats of ELISA for the detection of antibodies against swine vesicular disease (SVD) virus were developed. One of the tests described is a monoclonal antibody-based competitive ELISA (MAC-ELISA). In this test, specific antibodies in serum are detected due to their ability to compete with a neutralizing monoclonal antibody (MAb). The second is an indirect trapping ELISA which employs isotype-specific MAbs to detect swine IgG or IgM specific for SVD virus. The diagnostic sensitivity and specificity of the MAC-ELISA was studied on 5671 field sera of known origin, enabling the cut-off level to be defined. Using the MAC-ELISA, 100% of sera from infected pigs were found positive, whereas only 0.45% of negative sera gave a false-positive result. A positive correlation between MAC-ELISA and virus neutralizing titres was recorded for pig sera collected sequentially after experimental infections. The results from the isotype-specific ELISA revealed the dynamics of the antibody response to SVD virus in pigs. The first antibodies were detectable as early as 3 days after experimental infection. Up to the 10th day, demonstrable antibodies were exclusively of the IgM class. IgG developed later, between 11 and 14 days postinfection and remained at a plateaux level throughout the whole investigation period. The two tests satisfy different diagnostic requirements: the MAC-ELISA is useful as a screening test, the isotype-specific ELISA has potential application for the determination of stage of infection. Both tests benefit from the use of MAbs in terms of specificity and standardization and have advantages over the virus neutralization test.

Animals↗
PubMed1995

Antigenicity of the rabbit hemorrhagic disease virus studied by its reactivity with monoclonal antibodies.

A panel of anti-rabbit hemorrhagic disease virus (anti-RHDV) monoclonal antibodies was produced and characterized. The ability of the MAbs to recognize epitopes present on RHDV capsids, European brown hare syndrome virus capsids and RHDV subunits was determined. Preliminary results on the neutralizing capacity of the MAbs were obtained by in vivo protection experiments. The antigenic map of RHDV obtained by this study is consistent with the current models of the calicivirus structure.

Animals↗
PubMed1994

The structure and antigenicity of a type C foot-and-mouth disease virus.

BACKGROUND: Picornaviruses are responsible for a wide range of mammalian diseases and, in common with other RNA viruses, show considerable antigenic variation. Foot-and-mouth disease viruses (FMDVs) constitute one genus of the picornavirus family and are classified into seven serotypes, each of which shows considerable intratypic variation. This antigenic variation leads to continuing difficulties in controlling the disease. To date the structure of only one serotype, O, has been reported. RESULTS: The three-dimensional structure of a serotype C (isolate C-S8c1) FMDV, has been determined crystallographically at 3.5 A resolution. The main chain conformation of the virion is very similar to that of type O1 virus. The immunodominant G-H loop of VP1, the presumed site of cell attachment, is disordered in both types of virus indicating a functional role for flexibility of this region. There are significant changes in the structure of other antigenic loops and in some internal regions involved in protomer-protomer contacts, including the entire amino-terminal portion of VP2, described here for the first time for a picornavirus. Antigenic sites have been identified by genetic and peptide mapping methods, and located on the capsid. The data reveal a major new discontinuous antigenic site (site D) which is located near to the three-fold axis and involves residues of VP1, VP2 and VP3 which lie adjacent to each other on the capsid. CONCLUSION: In FMDV type C, amino acid substitutions seen in mutants that are resistant to neutralization by monoclonal antibodies (MAbs) map to predominantly surface-oriented residues with solvent-accessible side-chains not involved in interactions with other amino acids, whereas residues which are accessible but not substituted are found to be more frequently involved in protein-protein interactions. This provides a molecular interpretation for the repeated isolation of the same amino acid substitutions in MAb-resistant variants, an observation frequently made with RNA viruses. This first comparison of two FMDV serotypes shows how subtle changes at antigenic sites are sufficient to cause large changes in antigenic specificity between serotypes.

Amino Acid Sequence↗
PubMed1994

Genetic variation of foot-and-mouth disease virus from field outbreaks to laboratory isolation.

Foot-and-mouth disease virus (FMDV), by nature of its RNA genome, possesses a high rate of mutation during replication. This results in extensive genetic polymorphism of virus populations in nature. The emergence of FMDV variants during replication has been reported. Genetic changes in the viral capsid protein (VP1) gene can result in amino acid changes affecting the immunodominant epitopes of FMDV. The genetic heterogeneity of FMDV in the field and the antigenic variants observed after cell culture isolation has been investigated by PCR sequencing and reactivity with monoclonal antibodies. These methods were applied to viruses causing two different outbreaks of FMD before and after replication in cell culture and in the animal host. The VP1 region of the genome was amplified by PCR and sequenced to reveal variant sequences identified after passage and to determine their presence in the original field tissue. In one case, reactivity with monoclonal antibodies was lost after passage as a result of an amino acid change in the subpopulation. These findings suggest that host cells can select specific virus genetic and antigenic subpopulations during virus isolation and propagation.

Amino Acid Sequence↗
PubMed1994

Beta 2 glycoprotein I and placental anticoagulant protein I in placentae from patients with antiphospholipid syndrome.

OBJECTIVE: To investigate the immunohistological distribution of beta 2-glycoprotein I (beta 2 GPI) and placental anticoagulant protein I (PAP-I) in normal and pathological placentae of patients with antiphospholipid (aPL) antibody associated recurrent fetal loss. These proteins are 2 natural anticoagulants able to interfere with aPL antibody binding. METHODS: Placentae from 4 patients with primary antiphospholipid antibody syndrome (pAPS), from 2 patients with aPL negative systemic lupus erythematosus (SLE) and from 7 healthy women were studied. Cryostatic placental sections were tested by indirect immunofluorescence using polyclonal anti-PAP-I and anti-beta 2GPI antisera as well as purified IgG and anti-beta 2GPI monoclonal antibody. The same tissue sections were also tested by direct immunofluorescence with FITC-F(ab)2 goat antihuman IgG. RESULTS: We found that (a) the placental distribution of PAP-I was comparable in normal and pathological specimens; (b) on the contrary, increased beta 2GPI deposition was present on the trophoblast surfaces of placentae obtained from patients with persistent raised titers of aPL antibodies. Comparable IgG deposition on villi surface was also found in aPL positive but not in control placentae. CONCLUSION: Our data are consistent with the hypothesis that high titer aPL binds to a beta 2GPI phospholipid complex in placentae of women with recurrent fetal loss but that a quantitative deficiency of PAP-I does not play a pathogenetic role in aPL associated fetal loss.

Adult↗
PubMed1993

Improvements in the diagnosis of contagious bovine pleuropneumonia through the use of monoclonal antibodies.

Seventeen monoclonal antibodies (MAbs) raised against Mycoplasma mycoides ssp. mycoides small colony type (Mmm SC), the causative agent of contagious bovine pleuropneumonia (CBPP), were partially characterised. Six MAbs recognising a main protein of 70 kDa and showing reciprocal competition were found to be specific for Mmm SC, while the other MAbs showed different patterns of reactivity with Mycoplasma spp. within the mycoides "cluster". Sandwich enzyme-linked immunosorbent assay (ELISA) performed with different combinations of MAbs enabled the detection of Mycoplasma of the mycoides cluster in pathology samples and the specific identification of Mmm SC from the initial culture passages, overcoming the necessity for difficult and time-consuming biochemical and growth inhibition assays. A competitive ELISA based on Mmm SC-specific MAbs was able to measure anti-CBPP antibodies in cattle sera, and detected only the set of antibodies directed at Mmm SC-specific epitopes, thus avoiding the false-positive reactions occasionally observed with the complement fixation test.

Animals↗
PubMed1993

Accuracy of endoscopic ultrasonography in the diagnosis and staging of gastric cancer and lymphoma.

BACKGROUND: There is a need to assess the sensitivity, specificity, and predictive value of endoscopic ultrasonography (EUS) in the diagnosis and staging of gastric cancer and lymphoma. METHODS: A prospective study was performed on 86 patients with endoscopic gross appearance suspicious for cancer or lymphoma. Biopsies with endoscopic forceps were always carried out before EUS. All patients underwent laparotomy for final diagnosis, staging, and eventually treatment. The results of EUS were correlated with the histologic findings of the resected specimens, when possible, or with the surgical findings. There were 42 gastric cancers and 44 primary gastric lymphomas. RESULTS: EUS made a correct diagnosis of cancer in 35 of 42 patients, with a sensitivity of 83%. Positive predictability was 87%, specificity was 97%, and negative predictability was 96%. Diagnostic accuracy was 95%. In the evaluation of cancer depth invasion, EUS was correct in 91% of cases. EUS displayed perigastric metastatic lymph nodes in 14 of 25 patients, with a sensitivity of 56%. Positive predictive value was 93%, specificity was 93%, and negative predictive value was 54%. Diagnostic accuracy was 69%. EUS made a correct diagnosis of lymphoma in 39 of 44 patients, with a sensitivity of 89%. Positive predictability was 87%, specificity was 97%, and negative predictability was 97%. Diagnostic accuracy was 95%. In the evaluation of lymphoma depth invasion, EUS was correct in 92% of cases. EUS displayed metastatic perigastric lymph nodes in 8 of 18 patients, with a sensitivity of 44%. Positive predictability was 100%, specificity was 100%, and negative predictability was 72%. Diagnostic accuracy was 77%. CONCLUSIONS: From these data it appears that in these diseases EUS has demonstrated specific ultrasonographic features that allow correct diagnosis and staging in the majority of patients. In difficult cases EUS may help to achieve the correct diagnosis. EUS also appear to be a useful tool for staging of gastric cancer and lymphoma. It shows not only tumor depth and local spread but also the passage from a pathologic to a normal wall and lymph node metastasis. With this accurate noninvasive staging procedure, in the near future many patients will no longer undergo exploratory laparotomy for surgical staging. Thanks to EUS, the choice of conservative or surgical treatment can be strongly affected. In case of surgery, EUS can orient the kind of surgical approach. Moreover, the use of EUS for evaluation of therapy during follow-up will probably become of major importance.

Adult↗
PubMed1992

Complement-mediated in vitro bactericidal activity of monoclonal antibodies reactive with outer-surface-protein OspB of Borrelia burgdorferi.

Murine monoclonal antibodies (mAbs) were obtained against the outer-surface-protein OspA and OspB and against the 41-kDa flagellar antigen of Borrelia burgdorferi. The specificity of mAb was determined by the Western blotting technique and the surface association of the antigens was inferred by immunofluorescence of living bacteria. In an in vitro assay in the presence of complement, two mAbs reactive with the Ospa were able to kill borreliae, whereas several mAbs reactive with the OspA as well as with the 41-kDa flagellar protein were not.

Animals↗