Two different cell populations, high- (MARC-145) and low-permissive cell clones (L-1) to porcine reproductive and respiratory syndrome (PRRS) virus, were derived from MA-104 cell line (parent cell: P) by cell cloning. Maximum virus yields in MARC-145, P, and L-1 cell clones were 10(8.5), 10(3.5), and 10(2.5) tissue culture infective dose 50 (TCID50)/0.1 ml, respectively. The MARC-145 cell clone supported replication of all 11 different porcine reproductive and respiratory syndrome virus isolates that were tested. These results indicated that the MARC-145 cells will be useful for PRRS virus replication.

Various conditions were evaluated and modified to improve the sensitivity of the serum neutralization (SN) test for detecting antibody in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV). Higher SN titers were consistently obtained by the addition of 20% fresh swine serum to the virus diluent and by the use of a permissive cell clone (MARC-145) derived from the MA-104 cell line. Test sera used to assess the SN test were obtained from 2 groups of 3-week-old pigs infected intranasally with PRRSV (MN-1b). Using the modified method, SN antibody was first detected 9-11 days postinoculation (PI), with a peak evident at 11-21 days PI. The antibody subsequently declined, and a second peak was observed between 41 and 45 days PI. The first antibody peak was not observed and the SN antibody was only detectable between 32 and 41 days PI when the test was done with 20% heated swine serum or without supplemental swine serum. The SN antibody during 2-3 weeks PI was found to be sensitive to 2-mercaptoethanol or anti-swine IgM treatment. The SN antibody titers were high when homologous PRRSV isolate was used in the test but were markedly low for heterologous PRRSV isolates. No difference in antibody titers was observed when homologous and heterologous PRRSV isolates were tested by indirect fluorescent antibody assay. These results indicate that the modified SN method is useful in detecting earlier and higher PRRSV antibody and that it can differentiate among PRRSV isolates.

Abstract. An indirect fluorescent antibody (IFA) test was developed and standardized to detect and quantitate antibody for swine infertility and respiratory syndrome (SIRS) virus in swine sera. Test results were evaluated using sera of pigs infected both experimentally and naturally with SIRS virus. The IFA test used swine alveolar macrophage (SAM) monolayers prepared in 96-well microplates and infected with SIRS virus. The monolayers were incubated with test sera, washed, and stained with fluorescein isothiocyanate-labeled rabbit anti-swine IgG. After another wash step, the monolayers were examined under a fluorescent microscope. A noninfected SAM control well was included for each sample. The antibody titers for each serum sample were recorded as the highest serum dilutions with specific cytoplasmic fluorescence but no fluorescence in the control wells. To evaluate the test, sera of 4 6-week-old pigs that had been infected with SIRS virus, 2 contact pigs, and 13 experimentally infected sows were used. In the experimentally infected pigs, antibody was first detected at 7 days postexposure (PE) and peaked (1:256-1,024) between 11 and 21 days PE. All 13 sow sera were negative at time of infection but were positive (1:64-> 1: 1,024) at 14-26 days PE. Seven hundred twenty sera collected from 25 different swine farms with or without a history of SIRS were also tested. Of 344 sera from 15 swine farms with a clinical history of SIRS, 257 (74.7%) sera had IFA titers ≥ 1:4, whereas 371 (98.7%) of 376 sera from herds with no history of SIRS were negative. The present results indicate that the IFA is a useful test for the detection and quantitation of SIRS virus antibody in swine sera.Swine infertility and respiratory syndrome (SIRS) virus has recently been recognized as an important pathogen in the United States because the virus causes severe reproductive failure in pregnant sows and high mortality due to respiratory disease in young pigs. [1][2][3]8 A similar syndrome has been reported in several European countries, 4-7 and the causative agent, Lelystad virtus, 6 was identified. Antibody to the Lelystad virus has been detected by an immunoperoxidase monolayer assay (IPMA), 6 whereas a serum neutralization (SN) test was described for the detection of antibody to SIRS virus.2 The purpose of the present study was to develop a simple indirect fluorescent antibody (IFA) method for detection and quantitation of SIRS virus antibody in swine sera. The method was evaluated using sera of pigs experimentally infected with SIRS virus. Test results of field serum samples collected from 25 different farms with or without a clinical history of SIRS are also described.SIRS MN-lb isolate at the fifth passage level was prepared in SAM cells, and virus aliquots of 10 4.5 immunofluorescence infective dose 50 (IFID 50 )/ml were stored at -70 C. Materials and methodsPreparation of test plates. The SAM cells, either freshly prepared or stored at -70 C, were diluted at a concentration of 2 x 10 6 cells/ml in RPMI-1640 medium supplemented w...

Abstract. The American and European strains of porcine reproductive and respiratory syndrome (PRRS) virus were initially isolated in an established cell line (CL 2621) and porcine alveolar macrophages (PAM), respectively. Subsequent isolation of American strains of this virus in PAM has also been reported. To determine their relative sensitivity for virus isolation, both PAM and CL 2621 cells were inoculated with 98 tissue specimens and 73 serum samples from animals suspected of having PRRS. Four of the 98 tissue samples yielded virus in both cell types, whereas 7 samples were positive only in PAM and 4 samples only in CL 2621. Of the 73 serum samples tested, 18 were positive in PAM of which only 2 were positive in CL 2621. Additionally, 82 isolates obtained initially in CL 2621 were inoculated in PAM cells, and 18 strains isolated originally in PAM were inoculated in CL 2621. Of the 82 CL 2621 isolates, 25 could not be propagated on PAM. Of the 57 that replicated in PAM, as detected by a positive test on indirect fluorescent antibody test, only 28 produced cytopathic effects and 29 did not. Of the 18 PAM isolates, 5 did not grow on CL 2621. Although PAM were relatively more sensitive for virus isolation, their failure to support the growth of certain strains of PRRS virus indicates the existence of variants among PRRS virus strains, and both PAM and CL 2621 should be used for virus isolation from clinical samples. In addition, the sensitivity of these 2 cell types was compared for the detection of fluorescent antibodies to PRRS virus using 179 serum samples from PRRS-infected animals. The results were comparable in both cell systems.

Abstract. Severe clinical signs of swine infertility and respiratory syndrome (SIRS) of unknown cause were observed in several Minnesota swine farms between November 1990 and March 1991. Forty-five lung samples of weak pigs were collected from 13 swine farms, and virus isolation was attempted using swine alveolar macrophage (SAM) cultures. A cytopathic virus was isolated from 19 lung samples collected from 6 different farms. Four pregnant sows were infected intranasally with a tissue suspension from which virus was isolated, and 4 6-week-old pigs and 2 contact pigs were infected intranasally with 1 of the isolates. The 4 sows farrowed 12 stillborn and 32 normal pigs. Virus was recovered from 10 of 19 pigs examined. Infected 6-week-old pigs were clinically normal except for slightly elevated rectal temperatures and mild respiratory signs. No or mild interstitial pneumonic lesions were observed in inoculated pigs, but the lesion was obvious in the 2 contact pigs. Seroconversion was observed in sows and pigs as measured by indirect fluorescent antibody (IFA). Serologic identification of the isolates was carried out by IFA using reference serum prepared from an experimentally infected sow. A cytoplasmic fluorescence was observed on the SAM monolayers infected with each of the 19 different isolates. Fluorescence was also observed when the monolayers were tested with SIRS virus ATCC VR-2332-infected sow sera. Replication of the isolates was not affected in the medium containing 5-iodo-2'-deoxyuridine but was inhibited by treatment with ether. The isolates were relatively stable at 56 C and did not agglutinate with various erythrocytes tested. These results are similar to those of previous reports, and the isolates, designated as SIRS virus, were the cause of SIRS on the farms investigated.