Detection of economically important viruses in boar semen by quantitative RealTime PCR technology.

The objective of this study was to develop quantitative real-time polymerase chain reaction (ReTi-PCR) tests for the detection of five economically important viruses in swine semen namely, pseudorabies virus (PRV), classical swine fever virus (CSFV), foot-and-mouth disease virus (FMDV), swine vesicular disease virus (SVDV), and porcine reproductive and respiratory syndrome virus (PRRSV). Each ReTi-PCR test was validated for specificity, analytical sensitivity (detection limits), and experimental infection studies were performed to compare the conventional virus isolation methods with the newly developed ReTi-PCR tests. All five developed ReTi-PCR tests are very rapid compared to virus isolation, highly specific, and even more sensitive (lower detection limits) than conventional virus isolation methods for the detection of mentioned viruses in semen. In semen of experimentally infected boars, viruses were detected much earlier after infection and more frequently by ReTi-PCR tests than by virus isolations. The high throughput of these rapid ReTi-PCR tests makes it possible to screen large number of semen samples for the presence of viruses prior to insemination. This is a substantial advantage, in particular for boar semen the quality of which deteriorates quickly after storage. In general, the newly developed ReTi-PCR tests are valuable tools for the early, reliable and rapid detection of five economically important viruses, namely PRV, CSFV, FMDV, SVDV, and PRRSV in boar semen. These ReTi-PCR tests will improve the control of viral diseases transmitted via semen.

Assessment of replication and virulence of attenuated pseudorabies virus in swine.

A nonclinical study was conducted to characterize the replication behavior of a modified live gE-deleted pseudorabies virus (PRV MS+1) in swine and potential for reversion to virulence after animal passages. Two to 3 week-old weaned pigs, negative for PRV, were maintained in isolation and challenged by intranasal instillation. For the first passage, 6 pigs were given 1 mL of PRV MS+1 (10(7.3)TCID(50)/mL) and 2 were necropsied at 3, 4 and 5 days post-inoculation (PI). Brain and secondary lymphoid tissues were collected, homogenized and the supernatants individually pooled for virus isolation, and PRV was recovered from each sample. No clinical signs of PRV infection were observed, but each pig had a nasal swab suspect or positive for PRV. For the second passage, 5 pigs were given 1 mL of the homogenate of mixed tissues from 1 animal in the previous passage (PRV at 10(1.9) TCID(50)/mL). At 5 days PI, all pigs were necropsied, and PRV was not recovered from their tissue homogenates or nasal swabs, and no clinical signs were observed. During a second attempt at a second passage, tissue homogenates from all pigs in the first passage (PRV at approximately 10(1.7)TCID50(50)/mL) were pooled and used to inoculate 15 pigs with 2 mL for 3 consecutive days. Ten pigs were monitored for clinical signs and seroconversion through 21 days PI, and 5 pigs were necropsied at 5 days PI. No clinical signs or PRV antibodies were detected in the 10 monitored pigs, and no PRV was recovered from the homogenates or nasal swabs of the 5 necropsied pigs. Thus, no evidence of reversion to virulence was demonstrated in pigs given the attenuated PRV.

Quantitative assessment of genomic similarity from restriction fragment patterns.

A quantitative algorithm for comparing restriction fragment patterns (RFPs) was developed and used to estimate the genomic similarity of 18 isolates of pseudorabies virus of known origin. Variants of this algorithm using either untransformed or square-root-transformed differences between fragment sizes were investigated with regard to their ability to classify RFPs. Multidimensional scaling was used to represent spatially the genomic relatedness among samples, with 3 dimensions producing the most meaningful results. The square-root transformation provided more interpretable dimensions. The first dimension differentiated samples geographically, separating North American from European isolates. The second and third dimensions differentiated isolates with specific gene deletions (gE and gG, respectively) from those not having these deletions. Clusters of isolates were identified that were related either by collection from the same geographic area during a specific time period, or by laboratory intervention to create vaccines. These methods offer increased precision in the determination of genetic relatedness based on RFPs, and thus offer increased diagnostic accuracy for the determination of sources of infection.

Detection of virus or virus specific nucleic acid in foodstuff or bioproducts--hazards and risk assessment.

There are two possibilities for virus contamination of foodstuff and bioproducts of animal origin: i) the presence of endogenous virus as a result of an acute or subclinical infection of animal raw material used for food processing or ii) contamination of food in the course of processing or thereafter. The latter must be considered as the highest risk for human consumers since the viral contamination mostly is caused by virus shedding people and the transmitted viruses are obligate human pathogens. Food from animals consumed as raw material (e.g. oysters) is listed in a high risk category concerning viral contamination (e.g. hepatovirus). Virus contamination of bioproducts such as vaccines, blood products or biological material used in surgery and for transplantations also is more hazardous because the application of contaminating virus usually occurs by circumvention of the natural barrier systems of the body. Moreover, in many cases immunosuppressed people are treated with bioproducts. Due to an enclosing shield of high protein and lipid content in food and bioproducts viruses are well protected against physical and chemical influences, however most preparation procedures for food are destructive for viruses. The detection of pseudorabies virus and pestivirus in biological fluids was tested using polymerase chain reaction (PCR), reverse transcriptase (RT)-PCR and cell culture propagation. PCR is a powerful method to detect viral nucleic acid whereas the detection of infectious virus in cell cultures is more limited, e.g. due to protein and lipid destroying conditions. Virus contamination of bioproducts should be considered as a hazard no matter which method has been used for its detection. Examples are given about the contamination of cell lines and vaccines.