Characterization of PRRSV in clinical samples and the corresponding cell culture isolates.

Isolation of porcine reproductive and respiratory syndrome virus (PRRSV) in cell culture is a primary means of obtaining virus isolates for autogenous vaccine production and other applications. However, it has not been well characterized whether cell culture isolate and the virus in clinical sample are equivalent. This study compared PRRSV ORF5 sequences from 1024 clinical samples (995 PRRSV-2, 26 PRRSV-1, and three PRRSV-1 and PRRSV-2 PCR-positive) and their isolates in MARC-145 and/or ZMAC cells. For three PRRSV-1 and PRRSV-2 PCR-positive clinical samples, both PRRSV-1 and PRRSV-2 were isolated in ZMAC cells, whereas either PRRSV-1 or PRRSV-2, but not both, was isolated in MARC-145 cells, with isolate sequences matching the respective viruses in clinical samples. Twenty-six PRRSV-1 and most of 995 PRRSV-2 PCR-positive clinical samples had matching viral ORF5 sequences with their cell culture isolates. However, 14 out of 995 PRRSV-2 cases (1.4%) had nonmatching viral sequences between clinical samples and MARC-145 isolates, although viral sequences from clinical samples and ZMAC isolates matched. This is concerning because, if the MARC-145 isolate is directly used for autogenous vaccine production without sequencing confirmation against the virus in the clinical sample, it is possible that the produced autogenous vaccine does not include the desired wild-type virus strain found on the farm and instead contains vaccine-like virus. Vaccine-specific PCR and next-generation sequencing performed on six selected cases indicated presence of ≥2 PRRSV-2 strains (mixed infection) in such clinical samples. In summary, PRRSV ORF5 sequences from clinical samples and cell culture isolates matched each other for majority of the cases. However, PRRSV sequences between clinical sample and MARC-145 cell culture isolate could occasionally be different when the clinical sample contains ≥2 PRRSV-2 strains. Characterizing PRRSV sequences from clinical samples and cell culture isolates should be conducted before using isolates for producing autogenous vaccines or other applications.

Comparison of ZMAC and MARC-145 Cell Lines for Improving Porcine Reproductive and Respiratory Syndrome Virus Isolation from Clinical Samples.

The MARC-145 cell line is commonly used to isolate porcine reproductive and respiratory syndrome virus (PRRSV) for diagnostics, research, and vaccine production, but it yields frustratingly low success rates of virus isolation (VI). The ZMAC cell line, derived from porcine alveolar macrophages, has become available, but its utilization for PRRSV VI from clinical samples has not been evaluated. This study compared PRRSV VI results in ZMAC and MARC-145 cells from 375 clinical samples (including 104 lung, 140 serum, 90 oral fluid, and 41 processing fluid samples). The PRRSV VI success rate was very low in oral fluids and processing fluids regardless of whether ZMAC cells or MARC-145 cells were used. Success rates of PRRSV VI from lung and serum samples were significantly higher in ZMAC than in MARC-145 cells. Lung and serum samples with threshold cycle (CT ) values of <30 had better VI success. PRRSV-2 in genetic lineages 1 and 8 was isolated more successfully in ZMAC cells than in MARC-145 cells, whereas PRRSV-2 in genetic lineage 5 was isolated in the two cell lines with similar success rates. For samples with positive VI in both ZMAC and MARC-145 cells, 14 of 23 PRRSV-2 isolates had similar titers in the two cell lines. A total of 51 of 95 (53.7%) ZMAC-obtained PRRSV-2 or PRRSV-1 isolates grew in MARC-145 cells, and all 46 (100%) MARC-145-obtained isolates grew in ZMAC cells. In summary, ZMAC cells allow better isolation of a wide range of PRRSV field strains; however, not all of the ZMAC-obtained PRRSV isolates grow in MARC-145 cells. This report provides important guidelines to improve isolation of PRRSV from clinical samples for further characterization and/or for producing autogenous vaccines.

A porcine macrophage cell line that supports high levels of replication of OURT88/3, an attenuated strain of African swine fever virus.

The main target cells for African swine fever virus (ASFV) replication in pigs are of monocyte macrophage lineage and express markers typical of the intermediate to late stages of differentiation. The lack of a porcine cell line, which accurately represents these target cells, limits research on virus host interactions and the development of live-attenuated vaccine strains. We show here that the continuously growing, growth factor dependent ZMAC-4 porcine macrophage cell line is susceptible to infection with eight different field isolates of ASFV. Replication in ZMAC-4 cells occurred with similar kinetics and to similar high titres as in primary porcine bone marrow cells. In addition we showed that twelve passages of an attenuated strain of ASFV, OURT88/3, in ZMAC-4 cells did not reduce the ability of this virus to induce protection against challenge with virulent virus. Thus, the ZMAC-4 cells provide an alternative to primary cells for ASFV replication.