Early Serodiagnosis of Porcine Reproductive and Respiratory Syndrome Virus Infection of Pigs by Detection of Slow-Reacting and Complement-Requiring Neutralizing Antibodi.

Abstract

ABSTRACT. In order to evaluate and enhance the sensitivity of the neutralization (NT) test for detecting antibody in pigs infected with porcine reproductive and respiratory syndrome (PRRS) virus, the effect of altered incubation conditions and complement use on neutralizing (NT) antibody titer were investigated. Higher NT antibody titers were consistently obtained by addition of 20% guinea pig fresh serum to virus-serum mixtures in NT tests. Furthermore, the complement-requiring NT antibody titer increased in many serum samples when the virus-serum mixtures, rather than being incubated at 37 °C for 60 min, were incubated first at 4 °C for 48 hr and then with a complement at 37 °C for 60 min. The slow-reacting and complement-requiring NT antibody was detected as early as 8 days post-inoculation. It was detected in sera collected at 8 to 28 days post-inoculation and was sensitive to 2-mercaptoethanol treatment. Sera collected at 35 to 44 days post-inoculation contained 2-mercaptoethanol resistant NT antibodies. These results indicate that the modified NT test is useful for early serodiagnosis of PRRS virus infection through detection of higher NT antibody titers, and in detecting them earlier. MATERIALS AND METHODSCell culture: The MARC-145 cells were derived from a monkey kidney cell line (MA-104) [4]. The cells were grown in Eagle's minimum essential medium (MEM) containing 8% fetal calf serum. The maintenance medium was MEM containing 2% fetal calf serum and 50 µg/ml of gentamycin.Virus: The EDRD-1 strain of PRRS virus at the 10 to 13th passage level in MARC-145 cell cultures was used [7]. Culture fluid harvested from infected MARC-145 cell cultures was clarified by centrifugation and stored at 80°C. The infectivity assay was carried out in 96-well microplate cultures of MARC-145 cells. The inoculated cultures were examined for CPE after incubation at 37 °C for 5 days, and the 50% infectious dose (TCID 50 ) was calculated.Experimental infection of pigs: The method of experimental infection has been described previously [7]. Briefly, three 7-week-old pigs (No. 1-3) initially seronegative for the PRRS virus were infected intranasally with 2 ml of 10 6.4 TCID 50 /ml of the EDRD-1 strain of the PRRS virus. One pig (No. 4) served as an in-contact control. Serial serum samples were collected daily until 14 days post-inoculation, after which they were collected once weekly.NT test: The NT test was carried out in flat-bottomed 96-well microplates. In tests, serial 2-fold dilutions of the serum inactivated at 56°C for 30 min were made in the maintenance medium. In conventional NT tests, 120 µl of each dilution was mixed with an equal volume of Porcine reproductive and respiratory syndrome (PRRS) virus, family Arteriviridae, causes reproductive disturbance in sows and respiratory distress in piglets, and is prevalent worldwide [1,2,4,5,[7][8][9][10].The neutralization (NT) test, the immunoperioxidase monolayer assay (IPMA), the indirect immunofluorescence (IIF), and the enzyme-linked immunosorbent assay (ELISA) have all...

Evaluation of Enzyme-Linked Immunosorbent Assay (ELISA)and Immunofluorescent Antibody (IFA) Test for the Detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Antibody in Pigs from Conventional Farms.

“…S/P ratio calculation formula : S/P = [(absorbance value of sample in PRRS well) -(absorbance value of sample in NHC well)]/[(mean absorbance value of PC in PRRS well) -(mean absorbance value of PC in NHC well)] PC : positive serum control NHC : normal host cell Evaluation of non-coincident cases: Out of the sera showed non-coincident results in IFA and ELISA, available samples were tested by VNT and examined for PRRSV gene by the reverse transcription-polymerase chain reaction (RT-PCR) to evaluate which result is reliable. VNT was carried out according to the procedure of Takikawa et al [23]. RT-PCR was also performed by the method of Kono et al [14].…”

“…Therefore a highly sensitive and specific assay is needed. The IFA, the immunoperoxidase monolyer assay (IPMA), the virus neutralization test (VNT), and the haemaglutination inhibition test (HI) have been reported as antibody assays of PRRS virus (PRRSV) [1,7,8,13,23,27,29]. IFA has been most frequently used for PRRS in Japan.…”

Analysis of viral proteins of porcine reproductive and respiratory syndrome virus in host protection and early virus-cell interactions

“…Neutralizing antibodies develop slower and show lower titers than IFA antibodies (Frey etal., 1992). SVN test was modified to improve its sensitivity and can detect neutralizing antibodies earlier and higher (Jusa et al, 1996b, Takikawa et al, 1997, Yoon etal., 1994a. obtained higher neutralizing antibody titers consistently by the addition of 20% pig fresh serum to the diluted virus and by use of continuous cell line MARC-145.…”

Immunological significance of genetic variation in structural proteins and the genetic determinants for cross protection of Porcine Reproductive and Respiratory Syndrome (PRRS) virus

“…These sites are located between two putative transmembrane (TM) regions (65-130 and 170-190). The topology of this domain and C-terminus (191)(192)(193)(194)(195)(196)(197)(198)(199)(200)(201) (57) reported the importance of the last 7 residues at the C-terminus of ORF5 in the viability of PRRSV. It was, therefore, concluded that the C-terminus of ORF5 is exposed to the inside of the virion and interacts with other viral proteins to incorporate those proteins into the virion during virus assembly.…”