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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.

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.

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., Work at Centre de Investigació i Desenvolupament in Barcelona was supported by grants PB92-0707 and PB 95-0218 from DGICYT, that in Madrid was supported by grant PB 94-0034-C02-01 from DGICYT and Fundación Ramón Areces, and that at Universitat de Barcelona was supported by grants PB94-0845 and PB95-1131 from DGICYT. The Barcelona groups are part of CERBA from Generalitat de Catalunya

Pseudorabies (PR) (Aujesky’s disease) has been seen in several domestic species, such as pigs, ruminants, dogs, and horses. The laboratory diagnosis of PR has been investigated using various approaches. The most frequent have been immunofluorescence (IF) and immunoperoxidase methods, histopathologic investigation, tissue culture inoculation, and in situ hybridization. Immunoperoxidase staining can also be used to diagnose virus-induced nonsuppurative encephalitis, which is hard to differentiate histopathologically. Eighty monoclonal antibodies (MAbs) against PR virus (PRV) were produced at the Istituto Zooprofilattico to develop a competitive enzyme-linked immunosorbent assay to differentiate antibodies elicited by glycoprotein (G)F-negative strain vaccines from antibodies resulting from infection with wild type viruses and to develop a suitable diagnostic tool for the identification of PRV either directly from infected tissues or from inoculated cell cultures. Eight of the 80 MAbs reacted strongly in the IF test performed on frozen tissue sections (E. Brocchi, personal communication). The reactivity of these MAbs with paraffin-embedded sections was examined to evaluate immunoperoxidase staining for PRV diagnosis in routinely samples. Moreover, a retrospective study was conducted on animals submitted for necropsy because of clinical neurologic signs. The 8 MAbs specific for PRV (Table 1) were tested immunohistochemically with formalin-fixed, paraffin-embedded pig tonsils (for which PRV had been previously detected by IF on cryostatic sections). The following procedures were used for immunohistochemical staining. Five-micrometerthick paraffin-embedded sections were dewaxed in 2 changes of xilene for 15 minute each and hydrated through graded alcohols. All sections were treated with 3% hydrogen peroxide in methanol for 30-minutes at room temperature followed by several washes in Tris-buffered saline (TBS). Nonspecific antibody binding was blocked by 30 minutes of incubation with normal horse serum 1:75 diluted in TBS. As primary antibodies, ascitic fluid TBS dilutions (1:200, 1:400, 1:800, 1:1,000) of each MAb were incubated in 2 different ways, overnight at 4 C and 1 hour at 37 C. After MAb incubation, the sections were washed with TBS and sequentially incubated with biotinylated horse anti-mouse IgG 1: 200 diluted and streptoavidin peroxidase 1500 diluted for 30 minutes each, with rinses in TBS between the incubations. Diaminobenzidine was used as substrate of the peroxidase

The pathogenicity of two porcine encephalomyocarditis virus (EMCV) isolates for sows in gestation and young piglets was studied. One virus originated from a case of reproductive failure in pigs in Belgium and the other from a case of acute myocarditis in pigs in Greece. Sows in the mid-gestation period and one- to two-month old piglets were inoculated with each isolate. The molecular relationship between both isolates was studied by determining the nucleotide sequence located across the junction of the 1C and 1D capsid-coding genes. Antigenic analysis was performed using a panel of 35 monoclonal antibodies raised against an Italian field isolate of EMCV. All three approaches revealed differences between both isolates and also confirmed that there was no link between the two outbreaks of disease.

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 1D (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.

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.

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.

The causative agents of foot-and-mouth disease (FMD) are small icosahedral viruses of the Aphthovirus group within the Picornaviridae family. There is no evidence that these viruses infect cells of the immune system or otherwise interfere detrimentally with their function; additionally, it has not been possible to relate cytotoxicity reactions against virus-infected cells to the efficacy of the immune response against FMD virus infection. In contrast, there is a close association between FMD virus antibody and the protective immune response (10, 14, 15, 20, 24, 25, 29-32). Induction of this antibody is dependent on the structure of the viral antigenic sites (7-9, 11, 18) and on the concomitant presence of Th-lymphocyte epitopes (4, 5, 7, 8), although a Th-lymphocyte-independent response has been reported (2). Recent work by Piatti et al. (26) showed that the immune response induced by FMD virus was only Th-lymphocyte dependent when low doses of antigen were used. This latter work was performed in mice, and it is not certain that a similar situation would be found in cattle. As for the major effector immune defense, this relies on the interaction between antibody-virus complexes and the phagocytic cells of the reticuloendothelial system (17, 19).

Monoclonal antibodies (MAb) against an O, Suisse isolate of FMDV were used to identify epitopes on the virus particle and to determine their relative function. Six major antigenic sites containing one or more epitopes were identified using competition ELISA. An epitope relationship is proposed consisting of (i) a trypsin-sensitive sequential site, termed B 2 D 9 , from the codings for the MAb which reacted with it, which was associated with virus infectivity and is probably at or near to the cell-binding site of the virion; (ii) a trypsin-resistant, conformational site 1 C 6 4 C 9 MAb reaction at which also resulted in neutralization of virus infectivity; (iii) a second trypsin-resistant, conformational site 3C8, where again MAb reaction neutralised virus infectivity; (iv) a third trypsin-resistant, conformational site 6 C 3 2 G 5 , at which MAb-dependent neutralisation of virus infectivity was inefficient; (v) a site 3134, the expression of which was impaired but not destroyed by trypsin treatment, and was not related to virus infectivity; (vi) an internal site A8, which appears to be a “12S subunit-specific” site. This work clearly demonstrates for the first time that both trypsin-sensitive and trypsin-resistant neutralisable (infectivity-associated) sites exist on the FMDV particle, and only one of these can be related to the sequential site used to formulate current FMDV peptide vaccines.

A library of 27 murine monoclonal antibodies was obtained by using human liver and heart ferritins as immunogens. The specificity of the antibodies for the two ferritins and their subunits was studied with five different methods. The antibodies elicited by the liver ferritin bound preferentially the immunogen and were specific for the L subunit. Some antibodies elicited by the heart ferritin had characteristics similar to the anti-liver antibodies, other ones bound preferentially the heart over the liver ferritin and were specific for the H subunit. Only two antibodies were able to bind both ferritins and subunits. Some anti-H and anti-L chain antibodies were used to develop and compare four types of immunoassay to quantitate isoferritins. The results indicate the heart ferritin is immunologically more heterogeneous than liver, the H and L subunits having large immunological differences with few, if any, identical epitopes; and that that the architecture of the immunoassays have a strong influence on the crossreactivity of the antibodies with the two isoferritins, probably because H and L chains are not arranged randomly in the assembled protein.

Neutralizing monoclonal antibodies (nMAbs) elicited against foot-and-mouth disease virus (FMDV) of serotype C were assayed with field isolates and variant FMDVs using several immunoassays. Of a total of 36 nMAbs tested, 23 recognized capsid protein VP1 and distinguished at least 13 virion conformation-independent epitopes involved in neutralization of FMDV C. Eleven epitopes of FMDV C-S8c1 have been located in segments 138-156 or 192-209 of VP1 by quantifying the reactivity of nMAbs with synthetic peptides and with nMAb-resistant mutants of FMDV C-S8c1 carrying defined amino acid substitutions. The main antigenic site of FMDV C-S8c1 (VP1 residues 138 to 150) consists of multiple (at least 10), distinguishable, overlapping epitopes. Some amino acid replacements abolished one of the epitopes, whereas other replacements affected several epitopes in this region. The conservative substitution His(146)----Arg, found in many nMAb-resistant mutants analysed, abolished the reactivity of the virus with all nMAbs that recognized epitopes in the main antigenic site of FMDV C-S8c1. This indicates that a minimum genetic change can result in a highly amplified phenotypic effect, as regards the antigenicity of FMDV.

Using a monoclonal antibody (MAb) specific for the H7 influenza surface glycoproteins, a serological enzyme-linked immunosorbent assay (ELISA) test has been developed. This MAb was made using the low-pathogenicity (LP) avian influenza (AI) strain (BS2676/99) isolated in Italy during a recent outbreak. The test is able to detect H7 antibodies in avian sera. The H7 ELISA has a 99% concordance of results with the classical hemagglutination inhibition (HI) test.

A panel of monoclonal antibodies (mAbs) specific for the nucleocapsid (N) protein or the glycoprotein Gc of Schmallenberg virus (SBV), a novel member of the Simbu serogroup (genus Orthobunyavirus, family Bunyaviridae), was produced and used to analyze antigenic differences among members of this serogroup. Reactivity with various SBV-isolates and other Simbu serogroup viruses was assessed by an indirect immunofluorescence test and by immunoblotting. The Gc-specific mAbs detected different SBV isolates as well as two closely related members of the Simbu serogroup. In addition, one mAb showed a highly specific reactivity with the homologous SBV strain only. Based on their differing reactivity with different SBV-strains, these antibodies represent a valuable novel tool to rapidly determine the phenotype of new SBV isolates. In contrast, the N-specific mAbs showed a broad reactivity spectrum and detected not only all the tested SBV-isolates, but also several other viruses of the Simbu serogroup. One out of these mAbs even recognized all of the tested Simbu serogroup viruses in the indirect immunofluorescence assay. In order to further characterize the N-specific antibodies, PepScan analysis was performed and a specific epitope could be identified. In summary, the newly generated mAbs showed differing pan-Simbu virus-, pan-SBV- as well as SBV-isolate-specific reactivity patterns. Thus, they represent valuable tools for the development of novel antigen and antibody detection systems either specific for SBV or, in a broader approach, for the pan-Simbu serogroup diagnostics. Electronic supplementary material The online version of this article (doi:10.1186/s13567-015-0165-4) contains supplementary material, which is available to authorized users.