Immunodiffusion and neutralization studies on porcine adenoviruses

Precipitating antigens were prepared from porcine kidney cell cultures infected with the four approved serotypes of porcine adenoviruses (PAV) and from human amnion cell cultures infected with serotype 5 human adenovirus. For extracellular precipitating antigens (EPA) concentration by ammonium sulphate precipitation from cell culture fluids was used. Cell-associated precipitating antigens (CAPA) were extracted from cell sediments by repeated freezing and thawing. All antigens reacted alike and formed a single coalescent precipitin line of identity when tested against sera collected from a sow infected in the field or from weaners intranasally infected with serotypes 3 or 4 of PAV. In order to determine the optimal time of harvesting cell culture materials for the preparation of precipitating antigens, the kinetics of production and release of infectious virus and precipitating activity of PAV serotype 3 in porcine kidney cell cultures were studied. Precipitating activity first appeared with CAPA 24 h p.i. and 12 h later with EPA. Infectivity titers did not correlate with precipitating activities of EPA or CAPA beyond that stage. At the time the infectivity of EPA was decreasing, its precipitating titer continued to increase. The peaks of precipitating activities of CAPA and EPA were demonstrated at 96 and 144 h p.i., respectively. Three 7-week-old weaners with serum neutralizing antibodies against the four serotypes of PAV, but without detectable precipitating antibodies, were inoculated intranasally with serotype 3 of PAV. Serum samples collected 1 week p.i. showed precipitating activities and steep increases in neutralizing antibody titers against the homologous serotype 3 and the heterologous serotypes 1, 2 and 4 of PAV. The serum neutralizing antibody titers remained nearly constant over a period of 18 weeks p.i. while the intensity of the precipitating reaction decreased. Intranasal infection of the pigs with serotype 4 PAV induced heterotypic anamnestic neutralizing antibody response as well as an increase of the precipitating antibodies.     

Structural proteins of porcine enteroviruses

Analysis of the structural proteins of two strains (T80 and PE1) of cytopathogenic type 1 and two strains (V13 and T5) of cytopathogenic type II porcine enteroviruses by polyacrylamide gel electrophoresis, revealed polypeptide patterns which were generally similar to those described for other picornaviruses. However, one polypeptide (P5), of molecular weight 15 000 to 17 000, which was found in each strain of porcine enterovirus, has not been reported for other enteroviruses. A polypeptide (P4), of molecular weight 22 000, demonstrated in the type I strains, was lacking in the type II strains, and the molecular weight of the P2 polypeptide was lower in the type II strains than in the type I porcine enteroviruses.     

The isolation and characterization of porcine enteroviruses

Eleven enteroviruses representing four serotypes were isolated in a porcine kidney cell-line from swabs collected from a litter of apparently healthy piglets in New Zealand. One serotype produced type 2 cytopathic effect and was neutralized by type 8 porcine enterovirus antiserum. Of the remaining three serotypes which produced type 1 cytopathic effect, one was neutralized by type 1 antiserum and two were not neutralized at all by antisera types 1 to 8. Cross neutralization tests were not carried out. Nervous disease or reproductive disorders have not yet been associated with these viruses in New Zealand.     

Validation of rt-PCR assays for molecular characterization of porcine teschoviruses and enteroviruses

Porcine enteroviruses (PEVs) and teschoviruses (PTVs) are described as causative agents of neurological disorders, fertility disorders and dermal lesions of swine. Difficulties in the serological detection of these viruses may lead to a significant underestimation of infections with clinical symptoms. With the recent availability of genome sequence data for all the serotypes, molecular diagnosis is a possibility. The present study describes a new approach to molecular 'serotyping' of PTVs and PEV-B viruses, involving the amplification and sequencing of a genomic fragment of the VP1 coding region. A molecular characterization of Italian entero-teschovirus isolates was performed using a set of previously published and newly designed polymerase chain reaction primers. A total of 33 porcine isolates and 10 reference strains were analysed. Porcine enterovirus-B samples were first diagnosed as positive for enterovirus by amplification of the 5'-non-translated region. Samples were then typed by amplification and sequencing of a portion of the VP1 coding region. Porcine enterovirus-A and PTVs were detected by a published assay in the 5'-NC region that allows them to be differentiated according to the size of amplification product, using the same set of primers. For serotype characterization of PTV, we evaluated four different regions: the N terminus of the capsid protein VP2, the region encoding for RNA-dependent RNA polymerase, and the capsid VP1 and VP4 regions. The newly designed primers in the VP1 region was proved to be broad in range and suitable for serotype assessment and therefore constitute a useful diagnostic tool for molecular diagnosis of porcine teschovirus/enterovirus strains and for the study of molecular epidemiology and evolution of these viruses.     

Effect of actinomycin D on the cytopathology and replication of porcine enteroviruses

Actinomycin D inhibited the cytopathic effects (CPE) of type II strains of porcine enteroviruses in PK-15 cells but had no consistent effect on viral replication. The maximum inhibition of CPE was observed when the antibiotic was present during the early stages of infection. No inhibition of the CPE induced by type I strains was observed. A correlation was found between the activation of lysosomal acid phosphatase and morphological changes in the infected cells and the release of progeny virus. Diffuse enzymatic activity was more pronounced in cells infected with type II strains. The activation of lysosomal acid phosphatase by type II strains was inhibited by actinomycin D, while that of type I strains remained unaffected.     

Identification of group I porcine enteroviruses by monoclonal antibodies in cell culture

A panel of monoclonal antibodies (mAb) against porcine enteroviruses (PEV) was established. One of these mAbs reacts group-specifically with PEV of serotype group I in the indirect immunofluorescence assay (IIF). This mAb is very well suited for diagnosis of PEV infections. However, the mAb neither neutralizes virus nor does it react with virus particles in immuno electron microscopy (IEM). Another mAb is PEV-1 specific in IIF, neutralizes virus, and is suited for IEM. Both mAbs presumably recognize conformation-dependent epitopes of the virus.     

A serological comparison of 4 Japanese isolates of porcine enteroviruses with the international reference strains

The reference strains of four serotypes (J6, J8, J9 and J10) of porcine enterovirus isolated in Japan were compared with the international reference strains of 11 serotypes (W1 to W11) by cross neutralization tests. No cross reactions were observed between the two groups, although there were minor one-way crosses between W10 and J10, W11 and J9, and W4 and J9. This leads to our conclusion that all 4 Japanese serotypes can be newly added to 11 international serotypes. J9 virus produced type 1 of CPE (CPE I), J6 and J8 did CPE II, and J10 did CPE III. J10 grew in Vero, HeLa cells, and the primate cells, but J6, J8 and J9 did not.     

Cross reactions between porcine, bovine and ovine interleukin-2 preparations

Optimum conditions for the production of porcine interleukin-2 were found to include a delay of 24 hours before the addition of mitogen. Porcine and bovine interleukin-2 responded optimally in homologous systems whereas bovine interleukin-2 gave a better response in the ovine system than homologous ovine interleukin-2. Interleukin-2 produced from a continuous gibbon cell line reacted well with porcine, ovine and bovine T cell blasts indicating that it could act as a universal growth factor for T cell clones produced from these species.