PRRSV Strain Isolation and Characterization

Thirty-nine PRRSV strains were used in this study (Table 1). This set of strains included genotype-I (European) isolates from 1991 to 2006 of which some were retrieved from a viral collection (n = 15) and others were freshly isolated from frozen (-80 C) serum (n = 9) or lung tissue (n = 15) that have yielded positive results for PRRSV by RT-PCR. Freshly isolated viruses were from Spain and Portugal and archive viruses were from different countries of Western Europe. No epidemiological relationship was known to exist between the different isolates. Isolation was done in porcine alveolar macrophages (PAM) obtained from two healthy pigs free from all major diseases including PRRSV, pseudorabies virus and classical swine fever virus. Additionally, all PAM batches were tested for porcine circovirus type 2 (PCV2), hepatitis E virus and torque-tenovirus (TTV) according to previously described PCR protocols [14 (link)-16 (link)]. Viral stocks were also tested for mycoplasma by PCR. Viral stocks were produced from passages n = 2, n = 3 or n = 4 in PAM and, for each strain, batches of virus were larger enough to assure that the same batch could be used in all the experiments performed with that isolate, avoiding thus the use of different viral batches of the same strain for different experiments or replicas. Viral titrations were performed by inoculation of serial dilutions of viral stocks in PAM and readings were done by means of the immunoperoxidase monolayer assay using monoclonal antibodies for ORF5 (clon 3AH9, Ingenasa, Madrid, Spain) and ORF7 (clon 1CH5, Ingenasa, Madrid, Spain) using a method reported before with minor modifications [17 (link)].
In order to examine the need for virus viability for the induction of cytokines, three of the isolates were re-tested in parallel before and after inactivation by heat (60°C, 60 min). Complete inactivation was verified by inoculation of the heat-treated viral suspensions in PAM, which were examined at 72 h post-inoculation for the cytopathic effect and presence of PRRSV by IPMA. Untreated viable virus was used to assess the adequateness of the PAM batches for titrations.