Foot-and-Mouth Disease Virus Viroporin 2B Antagonizes RIG-I-Mediated Antiviral Effects by Inhibition of Its Protein Expression.

The role of retinoic acid-inducible gene I (RIG-I) in foot-and-mouth disease virus (FMDV)-infected cells remains unknown. Here, we showed that RIG-I inhibits FMDV replication in host cells. FMDV infection increased the transcription of RIG-I, while it decreased RIG-I protein expression. A detailed analysis revealed that FMDV leader proteinase (Lpro), as well as 3C proteinase (3Cpro) and 2B protein, decreased RIG-I protein expression. Lpro and 3Cpro are viral proteinases that can cleave various host proteins and are responsible for several of the viral polyprotein cleavages. However, for the first time, we observed 2B-induced reduction of host protein. Further studies showed that 2B-mediated reduction of RIG-I is specific to FMDV, but not other picornaviruses, including encephalomyocarditis virus, enterovirus 71, and coxsackievirus A16. Moreover, we found the decreased protein level of RIG-I is independent of the cleavage of eukaryotic translation initiation factor 4 gamma, the induction of cellular apoptosis, or the association of proteasome, lysosome, and caspase pathways. A direct interaction was observed between RIG-I and 2B. The carboxyl-terminal amino acids 105 to 114 and amino acids 135 to 144 of 2B were essential for the reduction of RIG-I, while residues 105 to 114 were required for the interaction. These data suggest the antiviral role of RIG-I against FMDV and a novel antagonistic mechanism of FMDV that is mediated by 2B protein. IMPORTANCE: This study demonstrated that RIG-I could suppress FMDV replication during virus infection. FMDV infection increased the transcriptional expression of RIG-I, while it decreased RIG-I protein expression. FMDV 2B protein interacted with RIG-I and induced reduction of RIG-I. 2B-induced reduction of RIG-I was independent of the induction of the cleavage of eukaryotic translation initiation factor 4 gamma or cellular apoptosis. In addition, proteasome, lysosome, and caspase pathways were not involved in this process. This study provides new insight into the immune evasion mediated by FMDV and identifies 2B as an antagonistic factor for FMDV to evade the antiviral response.

Lack of cross-species transmission of porcine endogenous retrovirus (PERV) to transplant recipients and abattoir workers in contact with pigs.

This study investigated the potential transmission of porcine endogenous retrovirus (PERV) to solid-organ transplant recipients and abattoir workers in contact with pigs. Blood samples were obtained from volunteer healthy blood donors (Group A; n=33); pig-breeding farmers who had undergone a liver transplant (Group B; n=14); and pig abattoir workers (Group C; n=49). A second blood sample was obtained 1 year after the first sample from 10 of the abattoir workers (Group D). Tests included investigation for PERV-DNA, PERV-RNA, pig-specific mitochondrial DNA, a quantitative detection of PERV nucleic acids, and antibodies to PERV by two different Western Blots. All polymerase chain reaction and Western Blots assays were negative for PERV or antibodies to PERV. Therefore, the risks of cross-species transmission of PERV appear to be negligible for immunocompetent individuals and allotransplant recipients, even if they are in close and repeated contact with live pigs or pig tissues.

[Express method for diagnostics of enzootic encephalomyelitis (Teschen disease) in pigs].

Results of creating the home ELISA test-system for diagnostics of enzootic encephalomyelitis (Teschen disease) has been presented. Biological components of diagnosticums has been created and described, reaction stages have been optimized. Sensitivity of the test-system for identification of antibodies to PTV-1 averaged 93%, specificity--100%. In the "sandwich"-variant the strains may be classified as serotype 1.

Induction of antigen-specific systemic and mucosal immune responses by feeding animals transgenic plants expressing the antigen.

A report from that the presence of lactogenic immunity in pigs protected suckling piglets from porcine epidemic diarrhea virus (PEDV) infection suggested that inducing mucosal immune responses in lactating pigs is an effective way of protecting swine from PEDV infection. In this study, we developed transgenic tobacco plants that express the antigen protein corresponding to the neutralizing epitope of PEDV spike protein, and tested whether feeding the plants to pigs induced an effective immune response against PEDV infection. First, we confirmed the immunogenicity of the plant-derived antigen by using a plaque reduction neutralization assay with serum obtained after injecting mice with protein extracted from the transgenic plants. Feeding the transgenic plants to mice induced both systemic and mucosal immune responses against the antigen. The induced antibodies inhibited virus infection in the plaque reduction neutralization assay. These results suggest that feeding animals transgenic plants carrying antigen genes is an effective strategy to induce protective immune responses against PEDV infection.

A prolonged outbreak of polioencephalomyelitis due to infection with a group I porcine enterovirus.

A commercial swineherd in Indiana experienced high death loss of nursery pigs with neurologic disorders for a prolonged period. Polioencephalomyelitis was the consistent histopathological lesion in affected animals. A porcine enterovirus (PEV) classified to group I was isolated from spinal cords and brains collected from the affected animals. The isolate (ISUVDL 200103183) was determined to belong to either serogroup 5 or 6 at the National Veterinary Services Laboratories, Ames, IA. No other significant viral or bacterial agents were isolated from or detected in the animals. A prospective longitudinal serological monitoring of pigs in the index herd for the PEV isolate revealed that colostrum-derived neutralizing antibodies to the virus rapidly declined, and by the age of 21 days the majority of piglets had no or minimal neutralizing antibody against the virus. Seroconversion to the virus then coincided with increased mortality in the herd. Results of diagnostic and cohort observations supported a diagnosis of PEV infection as the cause of the prolonged outbreak of "polio." Investigation into factors that may be contributing to the prolonged problem is currently in progress.

Porcine teschoviruses comprise at least eleven distinct serotypes: molecular and evolutionary aspects.

Nucleotide sequencing and phylogenetic analysis of 10 recognized prototype strains of the porcine enterovirus (PEV) cytopathic effect (CPE) group I reveals a close relationship of the viral genomes to the previously sequenced strain F65, supporting the concept of a reclassification of this virus group into a new picornavirus genus. Also, nucleotide sequences of the polyprotein-encoding genome region or the P1 region of 28 historic strains and recent field isolates were determined. The data suggest that several closely related but antigenically and molecular distinct serotypes constitute one species within the proposed genus Teschovirus. Based on sequence data and serological data, we propose a new serotype with strain Dresden as prototype. This hitherto unrecognized serotype is closely related to porcine teschovirus 1 (PTV-1, former PEV-1), but induces type-specific neutralizing antibodies. Sequencing of field isolates collected from animals presenting with neurological disorders prove that other serotypes than PTV-1 may also cause polioencephalomyelitis of swine.

[Serosurveillance of notifiable veterinary diseases in wild boar in the Netherlands]. / Sero-surveillance van veewetziekten bij wilde zwijnen in Nederland.

During the hunting season 1996-1999, blood samples were collected from wild boar shot in The Netherlands. Sera were screened for presence of antibodies against classical swine fever virus (CSFV), swine vesicular disease virus (SVDV), Aujeszky's disease virus (ADV), and Trichinella spiralis. The results indicate that CSFV, SVDV, and ADV are uncommon in the wild boar population. Therefore, it seems that CSFV, SVDV, and ADV infection in the wild boar population is not an important reservoir in The Netherlands. ADV and CSFV infections are endemic in the wild boar population in Germany. Since contact between the German and Dutch wild boar populations can not be excluded, continuation of the sero-surveillance system seems appropriate. In the decade before 1998, the wild boar population in The Netherlands seemed to be free of T. spiralis. Whether the finding, in the hunting season of 1998-1999, of a few wild boar with antibodies against T. spiralis is an artefact or not, should be investigated in further research.

Molecular cloning, expression and immunological analysis of the capsid precursor polypeptide (P1) from swine vesicular disease virus.

Swine vesicular disease virus (SVDV) is the aetiological agent of a highly contagious viral disease of pigs, whose symptoms are indistinguishable from those caused by foot-and-mouth disease virus (FMDV). The gene coding for the capsid protein precursor of SVDV (P1) from a recent spanish isolate (SPA/1/'93) was cloned and expressed in bacteria, and the antigenicity and immunogenicity of the recombinant product were evaluated. The recombinant P1 was recognised by antibodies against SVDV induced in pigs infected experimentally with different SVDV strains. Immunisation of swine with recombinant P1-induced SVDV-specific cellular and humoral immune responses. The implications of these results in SVD diagnostic as well as in vaccine development are discussed.

[Control of the identity of blood samples for the surveillance of research on swine vesicular disease and Aujeszky's disease]. / Identiteitscontrole van bloedmonsters voor de bewaking van het onderzoek op swine vesicular disease en de ziekte van aujeszky.

A surveillance programme for swine vesicular disease (SVD) and Aujeszky disease was set up in 1993 in the Netherlands. Blood samples are taken from pigs by local veterinarians to enable testing for the presence of antibodies against these viruses. A programme to guarantee the identity of pigs tested for these diseases has been in operation since late 1995. In this programme, pigs are identified on the basis of the DNA profiles of blood and hair samples. The hair samples are collected by the department of Animal Health Control. Analysis proves whether blood and hair samples are from the same animal. A total of 655 animals have been tested from 94 farms. In 38.6% of these farms differences were found between blood and hair samples.

Reduction of singleton reactors against swine vesicular disease virus by a combination of virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA.

Pigs which are serologically positive for swine vesicular disease virus (SVDV) but which show no clinical signs and for which there is neither a relevant history of the disease on the holding nor contact with a known outbreak are considered as singleton reactors. False positive serological results for an epizootic disease, like SVD, in a non-vaccinated population or in imported animals are of great concern to international trade. For the virus neutralisation test, the gold standard for SVD, singleton reactors are found at a level of 1-3/1000. Singleton reactors also occur when other serological testing methods are used. The number of animals finally considered as singleton reactors can be reduced considerably by performing three different serological tests (virus neutralisation test, monoclonal antibody-based competitive ELISA and isotype specific ELISA) on the same serum. A serological profile of the animal can be derived by analysing the results in greater detail. This procedure can reduce considerably the number of pig holdings on which a prohibition of movement and trade needs to be imposed without requiring analysis of supplementary samples.

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.

[Insight in SVD epidemiology. Specimen collection Regulation Farm Surveillance Animal Diseases]. / Inzicht in SVD-epidemiologie. Monstername Regeling Bedrijfscontrole Dierziekten.

Periodic (4-monthly) surveillance of pig herds for antibodies against swine vesicular disease (SVD) is obligatory in the Netherlands since 1993. Samples should be collected "at random' in as many compartments as possible, with a maximum of one pig per pen. This prerequisite for a sensitive surveillance will be elucidated using four examples of SVD outbreaks in the Netherlands.

Identification of the location of antigenic sites of swine vesicular disease virus with neutralization-resistant mutants.

Neutralization sites on swine vesicular disease virus (SVDV) have been identified by sequence analysis of neutralization-resistant mutants. Eight neutralizing monoclonal antibodies (MAbs) were produced and neutralization-resistant mutants were selected with the MAbs. Resistance of the mutants to neutralization was shown using the stab-neutralization method, and the results indicated the presence of five neutralization sites on the virus. The location of each site was identified from amino acid changes resulting from nucleotide substitutions in the mutants, and designated site 1 (residues 87 and 88 of VP1), 2a (residue 163 of VP2), 2b (residue 154 of VP2), 3a (residue 272 and 275 of VP1, 60 of VP3) and 3b (residues 70 and 233 of VP2, 73 and 76 of VP3). The locations of the amino acids substitutions at each site formed a cluster on a computer-simulated three-dimensional model of SVDV and were exposed to the surface of the virion.

Differential diagnosis and genetic analysis of the antigenically related swine vesicular disease virus and coxsackie viruses.

Monoclonal antibodies directed against an isolate of swine vesicular disease virus (SVDV), characterized by virus neutralization tests and competition assays, were used to compare SVDV isolates and isolates of the antigenically related Coxsackie viruses by ELISA. SVDV-specific reaction patterns and one specific for Coxsackie viruses were observed. This provided a method for distinguishing between these enteroviruses. In addition, RT-PCRs were undertaken with Coxsackie virus and SVDV genomes. Different product patterns were obtained which correlated with the genetic differences revealed by nucleotide sequence determination. RT-PCR distinguished between SVDV and Coxsackie viruses by pattern differences. Further SVDV-specific PCRs were carried out with clinical samples. Viral genomes were detected with a sensitivity equivalent to that of virus isolation in cell culture. Sequencing of the Coxsackie virus-derived 2A-coding PCR products resulted in a not previously described sequence of a B5 isolate and in SVDV-specific sequence of two Coxsackie virus A16 isolates. The differences of the isolates by ELISA and PCR reactivity, as well as the nucleotide sequence differences are consistent with the quasispecies concept of RNA viruses.

Validation of a screening liquid phase blocking ELISA for swine vesicular disease.

A direct liquid-phase blocking ELISA (LPBE) was developed for serological examination of swine vesicular disease (SVD). The sensitivity and specificity of the test were assessed on 272 and 365 sera collected on two farms where an outbreak had occurred. The specificity of the direct LPBE was higher than the specificity of the indirect LPBE. The European Community reference serum for SVD (RS 01-04-93), which has been adopted as the threshold for SVD serological examination in the European Community, had a mean titre of 2.19 in the neutralisation test. At a cut-off level of 2.0 in the neutralisation test, the sensitivity of the direct LPBE (screening at 1:432 final dilution) on the two farms was 90% and 99%, respectively. Based on these results, the screening dilution of the direct LPBE was adjusted to 1:160 final dilution, to obtain a sensitivity > or = 98% on both farms. Regression analyses showed a good correlation between the virus neutralisation test and the direct LPBE (r = 0.87). Compared to the indirect LPBE described before, the direct LPBE correlates better with the neutralisation test, has a higher specificity, and is more rapid. Because sera are tested in only one dilution, the test is highly suitable for the examination of large numbers of serum samples.

Grouping of porcine enteroviruses by indirect immunofluorescence and description of two new serotypes.

Porcine enteroviruses (PEV) types 1-11 were assigned to three serologic groups by indirect immunofluorescence (IIF). Serotype group I consists of PEV types 1-7 and 11 and is correlated with CPE-type I. Serotype group II is represented by PEV type 8, producing CPE-type II, while PEV types 9 and 10 are classified as serotype group III and cause CPE-type III. Three PEV isolates from the central nervous system of pigs with polioencephalomyelitis were assigned to serotype group I by IIF but to none of the established 11 serotypes by cross-neutralization. It is concluded that these isolates are representatives of two new PEV types for which the designation PEV12 and 13 is suggested.

Research on the presence of porcine enterovirus serotype 1 in north-eastern Italy.

The liquid-phase blocking sandwich ELISA, already developed for foot-and-mouth disease and swine vesicular disease serology, has been employed in pig sera for the antibody detection against porcine enterovirus serotype 1. ELISA-titers correlate positively with virus neutralization and their distribution show a high prevalence among the sampled pigs. The serological findings are confirmed by the isolation of a field strain of porcine enterovirus belonging to the serotype 1.

No serological evidence for the presence of swine vesicular disease virus in South Africa.

An indirect ELISA incorporating a protein A-peroxidase conjugate was developed for detecting antibodies to swine vesicular disease virus (SVDV) in pig sera. This test and a conventional virus neutralization test were found to be equally sensitive. A total of 2846 pig sera collected from various abattoirs in South Africa were tested using the indirect ELISA. No serological evidence of infection with SVDV in pigs in South Africa was found.