European serotype PRRSV vaccine protects against European serotype challenge whereas an American serotype vaccine does not.

Pigs were either vaccinated with an American serotype Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) vaccine or with a European serotype vaccine. A control group of was left unvaccinated. At four weeks after vaccination the PRRSV-specific antibody titres were determined and one third of each group was challenged with a Spanish, one third with a German and one third with a Dutch PRRSV wild type strain. The serological responses, measured at 4 weeks after vaccination, confirmed that both vaccines were of a different serotype. It was demonstrated that vaccination with an American serotype vaccine slightly reduced the amount of viraemia after challenge with European PRRSV wild type strains. Only after challenge with the Spanish PRRSV strain a moderate, and statistically significant, reduction in viraemia was observed. This is in contrast to vaccination with a European vaccine strain, where viraemia was completely suppressed after challenge with the German PRRSV isolate and almost completely suppressed after challenge with the Spanish and Dutch isolates.

Cell culture-grown putative bovine respiratory torovirus identified as a coronavirus.

A putative bovine respiratory torovirus (BRTV) was propagated in bovine fetal diploid lung and human colonic tumour cells, and fringed pleomorphic particles were detected in the culture supernatants by electron microscopy. Antisera directed against a bovine (Breda strain) and equine (Berne strain) torovirus failed to react with BRTV-infected cells in immunofluorescence assays and did not neutralise BRTV. No toroviral RNA was found in the supernatants of infected cells by means of a reverse transcriptase-polymerase chain reaction with torovirus-specific primers. On the other hand, bovine coronavirus-specific antisera and monoclonal antibodies did neutralise the cytopathic effects, and coronaviral antigen was detected in the cultures by immunofluorescence. Furthermore, bovine coronavirus RNA was detected in the supernatants of BRTV-infected cells after nucleic acid amplification. It is concluded that the cytopathic BRTV isolate is a coronavirus.

Effect on viraemia of an American and a European serotype PRRSV vaccine after challenge with European wild-type strains of the virus.

Three groups of 10 pigs were vaccinated with an American serotype porcine reproductive and respiratory syndrome virus (PRRSV) vaccine and three groups of 10 pigs were vaccinated with a European serotype PRRSV vaccine. A control group of 12 pigs was left unvaccinated. Four weeks after vaccination the PRRSV-specific antibody titres were determined and each group was challenged with either a Spanish, German or Dutch PRRSV wild-type strain. The serological responses four weeks after vaccination confirmed that the two vaccines were of different serotypes. Vaccination with the American serotype vaccine hardly reduced the level of viraemia after challenge with the European PRRSV wild-type strains, and only after challenge with the Spanish PRRSV strain was a moderate, statistically significant reduction in viraemia observed. In contrast, after vaccination with the European serotype vaccine, viraemia was completely suppressed after challenge with the German PRRSV isolate and almost completely suppressed after challenge with the Spanish and Dutch PRRSV isolates.

Porcine reproductive and respiratory syndrome virus (PRRSV): monoclonal antibodies detect common epitopes on two viral proteins of European and U.S. isolates.

Sixteen hybridoma cell lines secreting monoclonal antibodies (mAbs) directed against two dutch isolates of the causative virus of Porcine Reproductive and Respiratory Syndrome (PRRSV) were produced. The hybridoma cells resulted from fusions of SP2/0 myeloma cells with splenocytes of STU mice immunized with purified PRRSV after induction of immunotolerance against host cell constituents. Screening of supernatant fluids was performed by an indirect immunofluorescence assay on PRRSV-infected porcine alveolar macrophages. Immunoblotting studies revealed that the mAbs had different protein specificities. One mAb reacted with a viral 15 kD protein, eleven were directed against a 40-50 kD protein, and four against a 30-40 kD protein. The mAb against the 15 kD putative nucleocapsid protein as well as five mAbs against the 40-50 kD protein recognized epitopes on these proteins which are conserved in various European and U.S. isolates of PRRSV.

A mouse model for testing the pathogenicity of equine herpes virus-1 strains.

A mouse model was developed for testing the pathogenicity of equine herpes virus-1 (EHV-1) strains. The model was validated with EHV-1 strains that are known to be of a low or high pathogenicity in horses. From all parameters tested, the safety index, which was calculated from the body weights of the mice after infection, proved to be the best predictive parameter. When this parameter was used, good and reliable correlations were found with the pathogenicity of the EHV-1 strains in horses. This method enabled the differentiation between the two experimental EHV-1 strains whose genetic backgrounds were supposedly equal.

Detection of porcine reproductive respiratory syndrome virus by the polymerase chain reaction.

A polymerase chain reaction (PCR) is described for the detection of the porcine reproductive respiratory syndrome virus (PRRSV). Using the PCR we were able to detect about 30 infectious units of PRRSV resolved in tissue culture medium or sperm. For the detection of PRRSV in sperm the PCR is 10-times more sensitive than culturing in alveolar macrophages. For the detection of PRRSV, PCR provides a good alternative to cell culture methods.

Molecular characterization of porcine reproductive and respiratory syndrome virus, a member of the arterivirus group.

Porcine reproductive and respiratory syndrome virus (PRRSV)-specific cDNA clones spanning the 3' terminal 5 kb of the genomic RNA were isolated, sequenced, and used as probes for identification of PRRSV-specific RNAs. The PRRSV genome is a positive-stranded polyadenylated RNA of about 15 kb. In infected cells, a 3' coterminal nested set of six major subgenomic mRNAs could be demonstrated. Within the 3' terminal 3.5 kb of the PRRSV genome, six overlapping reading frames (ORFs) were identified, each most likely expressed by one of the subgenomic mRNAs. Amino acid sequence comparisons revealed that the most 3' terminal ORF (ORF7) encodes the PRRSV nucleocapsid protein with a calculated molecular weight of 14 kDa. It displays 44.8% amino acid identity with the capsid protein of lactate dehydrogenase-elevating virus (LDV) and 23.6% with that of equine arteritis virus (EAV). The product of ORF6, the second 3' terminal ORF, represents a putative membrane protein and exhibits 53.2 and 27.2% amino acid identity with the corresponding LDV and EAV polypeptides, respectively. Similar to EAV, ORFs 2 through 5 might encode glycosylated viral proteins. The polypeptide deduced from the most 5' ORF (ORF1b) contains two conserved domains common to EAV and coronavirus polymerases. Genome organization, strategy of gene expression, and the sequence of deduced proteins show that PRRSV belongs to the Arterivirus group of viruses.

Detection of proviral DNA and viral RNA in various tissues early after avian leukosis virus infection.

Using molecular biological techniques, a study was made of the tissue tropism of avian leukosis virus (ALV) early after infection. Two strains of chickens, one with and the other without endogenous viral genes, were infected with ALV of subgroup A immediately after hatching; specimens of nine tissues and blood samples were analyzed at various times thereafter. A polymerase chain reaction (PCR), specific to ALV subgroup A, was used to detect proviral DNA and viral RNA. In situ hybridization was used to confirm the presence of proviral DNA in tissue samples and to calibrate the PCR. The pattern of detection of proviral DNA and of ALV-RNA in the various tissues was similar for both chicken strains. At 2 weeks of age, ALV-RNA was demonstrated in all tissues tested: bursa of Fabricius, thymus, bone marrow, proventriculus, liver, spleen, kidney, muscle, gonads, and blood samples, and at 4 weeks of age all tissues contained proviral DNA. No tropism for a specific tissue was observed early after an ALV infection.

A two-dimensional 1H-NMR study on Megasphaera elsdenii flavodoxin in the oxidized state and some comparisons with the two-electron-reduced state.

Assignments for the 137 amino acid residues of oxidized Megasphaera elsdenii flavodoxin have been made using the sequential resonance assignment procedure. Great benefit was experienced from assignments of the fully reduced protein. The secondary and tertiary structures of the typical alpha/beta protein remain virtually identical on going from the oxidized to the two-electron-reduced state as judged from two-dimensional NOE spectroscopy. However, functionally important conformation changes in the flavin-binding region do occur on reduction. Considerable reduction-state-dependent chemical shift variations of protons in the immediate vicinity of the isoalloxazine moiety take place. From analysis of these shifts, it can be concluded that ring current effects of the pyrazine part of the flavin diminish on two-electron reduction.

[Transgenic chickens]. / Transgene kippen.

Transgenic mice are produced by retroviral insertion, micro-injection in the early embryo, and recently by transfection of embryonic stem cells. Transgenic chickens were only made by retroviral vectors based on avian leukemia virus (ALV) and reticuloendotheliosis (REV) genomes. A replication-defective retroviral vector is preferentially used because these do not induce infectious virus. Since chickens are lacking endogenous REV sequences, a replication defective REV vector is most useful for practical application. Transgenic disease resistance is most likely obtained by blocking of viral receptors. By this approach recently transgenics with resistance against ALV infection were made at the Regional Poultry Disease Laboratory in East Lansing. Inhibition of virus replication by antisense DNA, which is complementary to viral mRNA, is promising for the future. Considerable research efforts still have to be made, however. Production of biomedical proteins will most likely be the first practical use of transgenic chickens. For the time being, vaccines will be used for the control of infectious diseases. The current live-virus vaccines will be replaced by inactivated (sub-unit) vaccines and thereafter by recombinant DNA vaccines based on viral vectors.