Sequence analysis of porcine reproductive and respiratory syndrome virus of the American type collected from Danish swine herds.

Vaccine-like viruses of American type of porcine reproductive and respiratory syndrome virus (PRRSV) were detected in serum samples by RT-PCR. The viruses were analysed by nucleotide sequencing of the genomic region encoding open reading frames 2 to 7. During the ongoing study of Danish isolates of PRRSV by means of nucleotide sequencing, RT-PCR reactions and subsequent nucleotide sequencing showed the presence of American type PRRSV in Danish breeding herds. Most likely, these atypical viruses originated from boars vaccinated with live vaccine of American type (MLV RespPRRS), which were taken to artificial insemination centres and there brought together with unvaccinated boars already at the centres. The nucleotide sequences of three Danish viruses of American type PRRSV were compared to those of known PRRSV isolates. The nucleotide sequence identities of the atypical Danish isolates were between 99.2-99.5% to the vaccine virus RespPRRS and 99.0-99.3% to VR2332 which are the parental virus to the vaccine virus. Phylogenetic analysis including field isolates of American type supports the conclusion that the introduction of American type PRRSV in Denmark was due to spread of vaccine virus.

Differentiation of infection from vaccination in foot-and-mouth disease by the detection of antibodies to the non-structural proteins 3D, 3AB and 3ABC in ELISA using antigens expressed in baculovirus.

The baculovirus expression system was found to be efficient at expressing the 3D, the 3AB and the 3ABC non-structural proteins (NSP) of foot-and-mouth disease virus (FMDV) as antigens recognised by immune sera in ELISA. ELISA's using 3D, 3AB and 3ABC detected antibodies from day 8 and 10 after experimental infection of susceptible cattle and sheep and cattle remained seropositive for more than 395 days. The ELISA's detected antibodies against any of the seven serotypes of FMDV. The 3D ELISA was specific and precise and as sensitive as established ELISA's which measure antibody to structural proteins. The assay may be used as a resource saving alternative to established ELISA's for the detection of antibodies against any of the seven serotypes. The 3AB and the 3ABC ELISA were also specific and precise. FMDV infected cattle could be differentiated from those that had been merely vaccinated as they gave a positive result in both the 3AB and the 3ABC ELISA's. Two cattle that had been both vaccinated and infected also gave positive results in both tests, suggesting that the 3AB and 3ABC ELISA's, but not the 3D ELISA might represent a reliable means of detecting infection in a vaccinated population.

Reintroduction of bovine herpes virus type 1 into Danish cattle herds during the period 1991-1995: a review of the investigations in the infected herds.

In Denmark a programme for the systematic eradication of bovine herpes virus 1 (BHV-1) was completed during the years 1984 to 1991, but outbreaks due to new introductions of BHV-1 were seen. Between January 1991 and May 1994, 22 herds became infected with BHV-1, all located closely to the German border. In 1995, 61 herds were detected BHV-1 antibody positive, but they were situated in many different parts of Denmark. In order to find the source of infection owners of infected herds were interviewed, and restriction fragment pattern analysis (RFP-analysis) was performed on virus isolates from the herds with clinical outbreaks. Isolates from clinical outbreaks up to 1995 were identified as a Cooper-like strain, while 2 of those in 1995 had characteristics of a "new" strain, which had never before been identified in Denmark or elsewhere in Europe. In the described situation different transmission routes for virus seemed possible. One being a sporadic introduction of virus due to accidental contact with infected cattle near the German border or maybe due to an airborne transmission of virus over longer distance. The other, presumably a result of import of an infected animal despite the national regulations. The latter, due to an extensive trade pattern, resulted in the introduction of infected cattle into 51 BHV-1 seronegative cattle herds.

Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes.

Following the recent use of a live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in Denmark, both American (vaccine) and European-type PRRSV now coexist in Danish herds. This situation highlighted a requirement for supplementary tests for precise virus-typing. As a result, we developed a RT-PCR assay able to detect as well as type PRRSV. To provide maximal sequence information, complete viral open reading frames (ORFs 5 and 7) were targeted for amplification. The RT-PCR test was able to amplify complete PRRSV ORFs from complex materials such as boar semen containing as little as 1 TCID50 ml(-1) of PRRSV. Typing of viruses was accomplished by any one of three strategies: (i) use of type-specific PCR primers, (ii) size determination of ORF 7 amplicons, (iii) DNA sequencing. All three typing strategies showed complete concordance with the currently used method of typing with monoclonal antibodies (MAbs) when used on a panel of PRRSV field isolates covering the period 1992-1997. The ORF 7-based test had particularly desirable characteristics, namely, highly sensitive detection of PRRSV without apparent type bias, typing of the detected virus, discrimination between pure and mixed virus populations, and semi-quantitative assessment of type ratios in mixed populations, all in a single PCR reaction. In addition, the obtained sequence data were used to predict two simple and rapid strategies (single-enzyme restriction length polymorphy analysis and oligonucleotide hybridization) for confirmation of the specificity of ORF 7 RT-PCR reactions. As such, the RT-PCR assay provides a new, powerful diagnostic tool to study the population dynamics between present and emerging PRRSV-types.

Detection of antibodies against porcine parvovirus nonstructural protein NS1 may distinguish between vaccinated and infected pigs.

The humoral antibody response against the nonstructural protein NS1 and the structural protein VP2 of porcine parvovirus (PPV) was evaluated by immuno-peroxidase test (IPT) and enzyme linked immuno sorbent assay (ELISA) using recombinant PPV antigens. The coding sequence for NS1 and VP2 was inserted into the baculovirus. Autographa californica nuclear polyhedrosis virus (AcNPV) genome resulting in two recombinant baculoviruses AcNPV-NS1 and AcNPV-VP2, respectively. Sf9 cells (Spodoptora frugidiperda) inoculated with AcNPV-NS1 producing recombinant nonstructural protein (rNS1) and AcNPV-VP2 producing recombinant virion protein (rVP2) were used in IPT and ELISA to analyse serum antibodies. Pigs vaccinated with an inactivated whole virus vaccine and experimentally infected pigs were studied. Significant titers against rVP2 were obtained in both vaccinated and infected pigs. Specific antibodies against rNS1 could only be detected in infected pigs and NS1 may in this way allow the specific detection of infected animals. Analysis of serum samples collected up to 18 days post infection (p.i.) from four pigs experimentally infected with PPV showed that antibodies against rNS1 and rVP2 could in all cases be detected on day 9 p.i. Two individual pigs were inoculated twice with PPV and the antibody response was followed 89 days after second inoculation. Serum antibodies against both rVP2 and rNS1 could be detected for this period of time.

A contribution to the systematization of bovine herpesvirus 1 based on genomic mapping by restriction fragment pattern analysis.

Fourteen isolates of bovine herpesvirus 1 (BHV-1) found representative of more than 100 isolates studied, were compared by restriction fragment pattern analyses and molecularly characterized. A number of evolutionary links between the variants originally associated with infectious bovine rhinotracheitis and the variants originally associated with infectious pustular vulvovaginitis were identified. These findings, as well as the lack of any correlation between genome type and clinical manifestation, confirm that there is no phylogenetic basis for a distinction between groups of strains associated with genital and respiratory disease. Two attenuated vaccine strains can be identified as deviating from field isolates.