A. Solana

Phylogenetic relationships of European strains of porcine reproductive and respiratory syndrome virus (PRRSV) inferred from DNA sequences of putative ORF-5 and ORF-7 genes

The complete ORF-5 gene and a fragment of the ORF-7 gene from 14 different European porcine reproductive and respiratory syndrome virus (PRRSV) isolates were amplified by reverse transcriptase polymerase chain reaction (RT-PCR), and their nucleotide sequences were determined. The ORF-7 gene displayed nucleotide and amino acid identities of 94.1-99.6% and 95.3-100% among isolates from different countries. The ORF-5 gene showed higher nucleotide (87.1-99.2% identity) and amino acid (-88% identity) variability. The resulting sequences were aligned with other European and North American PRRSV strains and phylogenetic relationships among these strains were established by the maximum parsimony method. The phylogenetic trees inferred from both genes were in agreement and showed that European and North American PRRSV strains clearly represent two different genotypes. According to both trees, there is a perfect correlation between strains and the countries in which they were isolated. Additionally, the phylogenetic position of European and North American PRRSV strains within the recently proposed family Arteriviridae was also analyzed.

Direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus by reverse polymerase chain reaction (RT-PCR)

SummaryA method for direct detection of the porcine reproductive and respiratory syndrome (PRRS) virus was developed, based on reverse transcription of the viral RNA coupled to DNA amplification by polymerase chain reaction. A set of primers was designed from Lelystad virus sequence within ORF 7 encoding nucleocapsid protein. From seven Spanish field isolated strains the 312 bp amplified fragment was cloned and sequenced. Alignment with Lelystad virus sequence revealed a 96–97% homology. A maximum sensitivity of 6.7 TCID50 was achieved with the reported procedure in experimentally infected swine alveolar macrophages cultures. The sensitivity obtained in crude clinical samples from experimentally infected 3-weeks old pigs was approximately 102 TCID50. High specificity for the PRRS virus was demonstrated for the method, as none of the seven common swine virus assayed rendered DNA amplification product.

Rapid and high sensitivity test for direct detection of bovine herpesvirus — 1 genome in clinical samples

A procedure for direct detection of the BHV-1 genome in clinical samples, including semen, was developed. The method is based on the PCR amplification of a highly conserved DNA fragment within the glycoprotein gI sequence of the virus (323 bp between nt 1491 to nt 1814). The method is rapid and highly specific for all 27 subtypes assayed, which are included in the clinical and genetically different groups of BHV-1. The viral origin of the PCR product was assessed by Southern hybridization, with an internal probe. The method for DNA isolation from clinical samples included a fast extraction procedure with Chelex 100 resin allowing the loading of larger amounts of DNA in the PCR and in turn increasing the sensitivity of the method of detection. The level of sensitivity achieved by PCR was in the range of 1 TCID(50). This PCR assay may be an useful tool for BHV-1 monitoring in semen banks at low cost.

Semen changes in boars after experimental infection with porcine reproductive and respiratory syndrome (PRRS) virus

Eleven boars seronegative to porcine reproductive and respiratory syndrome virus (PRRSV) were trained for semen collection: five boars were inoculated intranasally with 6 x 10(6)TCID(50)/ml of PRRSV (Group A); four boars were inoculated intranasally with 6 x 10(4)TCID(50)/ml (Group B); and two boars were used as uninfected control (Group C). Semen samples were collected at 7-d intervals from 49 d prior to experimental inoculation with PRRSV to 70 d after inoculation, and were examined for sperm volume, sperm concentration, sperm morphology, sperm motility and for the presence of PRRSV. The infection in boars was demonstrated by the reisolation of PRRSV from the serum of all inoculated boars. Rectal temperatures and general health of the boars were clinically normal throughout the trial. Differences were observed in the quality of semen collected from boars after experimental infection with PRRSV. This infection induced a significant decrease in sperm motility and in spermatozoa with normal acrosomes. Of the semen samples tested for virus isolation in swine alveolar macrophages PRRSV was only isolated in 1 boar from Group B. The virus was detected in an additional semen sample in Group A by the production of an antibody titer in a biological assay. All attempts to detect PRRSV by RT-PCR in semen samples were unsuccessful. Nevertheless, from our study it is possible to suggest that the PRRSV can occasionally be transmitted in the semen during the initial phase of the disease.

Exposure of gilts in early gestation to porcine reproductive and respiratory syndrome virus

Twenty-five gilts without measurable serum antibody titres to porcine reproductive and respiratory syndrome virus (PRRsv) were identified and 16 were inoculated with PRRSV at seven, 14 or 21 days of gestation and killed 20 to 22 days later to determine the effect of the virus on their embryos. The remaining nine gilts were not exposed to PRRSV, but were killed at the same stages of gestation. The gults were observed for clinical signs of infection and the gilts and their embryos were tested for PRRv and homologous antibodies. The infection was demonstrated by the re-isolation of the virus and its detection by the reverse transcriptase polymerase chain reaction in serum and other tissue samples from the inoculated gilts, and also by seroconversion. However, the gilts remained healthy throughout the study, except for one which was depressed and anorexic for two days. Two of the litters from the gilts challenged with PRRSV on day 14 of gestation contained one and three infected live embryos; the other embryos from these two litters did not contain detectable virus, although most of the embryos in one of the litters were dead. The other nine litters from the gilts challenged with PRRSV and the control litters, showed no evidence of infection.

Rapid and sensitive detection of the bovine viral diarrhea virus genome in semen

A reverse transcription-polymerase chain reaction (RT-PCR) procedure was developed for the detection of bovine viral diarrhea virus (BVDV) in cell culture supernatant and in bovine semen. Several sets of primers, PCR conditions and extraction methods were examined to optimize the procedure. A set of primers designed to amplify a highly conserved portion of the p80 gene from BVDV (corresponding to NADL strain sequence from bp 6668 to 7107), was demonstrated to be the most effective. These oligonucleotide primers consistently amplify a 440-bp fragment from several non-cytopathic and cytopathic biotypes of BVDV. The viral origin of the PCR products was assessed by sequencing. The introduction of a Sephacryl S-400 chromatography step to remove seminal inhibitors prior to RNA extraction permitted RT-PCR detection of BVDV in raw and extended semen samples. A maximum sensitivity of 0.4 TCID50 was achieved with this method using RNA extracted from tissue supernatants. This RT-PCR assay may be a useful tool for the detection of BVDV in semen of persistently infected bulls.

Effect of porcine reproductive and respiratory syndrome virus (PRRSV) on development of porcine fertilizedova in vitro

Fertilized pig ova, collected from 14 estrus-synchronized gilts 32 h after presumed ovulation were microinjected or cultured for 72 h in Beltsville Embryo Culture Medium-3 with or without Porcine Reproductive and Respiratory Syndrome Virus (PRRSV). To detect virus in the samples, virus isolation of swine alveolar macrophage, Reverse Transcriptase Polymerase Chain Reaction and Fluorescent Antibody techniques were employed. Microinjection or incubation of embryos with PRRSV did not significantly inhibit development of the porcine embryos in vitro when compared with that of controls (P = 0.75 and P = 0.14, respectively). Although either 10 to 20 TCID50 were microinjected or large concentrations of virus were used for embryo exposure by incubation, PRRSV was not detected in association with the embryos. It is concluded based on the experiments reported here that 4- to 16-cell stage pig embryos are not susceptible to productive infection with PRRSV in vitro.