NC_OLD229: Porcine Reproductive And Respiratory Syndrome (PRRS): Mechanisms Of Disease And Methods For The Detection, Protection And Elimination of the PRRS Virus
Annual/Termination Reports (SAES-422): [12/06/2002] [02/03/2004]
Date of Annual Report: 12/06/2002
Report Information:
Participants:
URL: Copy of participant list
Brief Summary of Minutes of Annual Meeting:
URL: Copy of minutes
Accomplishments:
Abbreviated.Objective 1. MN, with the Danish Veterinary Institute, produced an infectious clone of strain VR-2332. Cytopathic effect (CPE) was seen only after several passages in MARC145 cells. MO, focusing on different potential pitfalls as compared to MN, cloned isolate NADC8. Transfections gave viral growth after 2-3 blind passages. Transfection supernatants injected into naove pigs yielded weakly positive serum, but none of the pigs seroconverted. ND and SD, with Guelph University (Canada), generated a clone of SD23983 that was not infectious. Clones were exchanged and are being used to generate chimeras in an effort to create an infectious clone. Nonstructural protein (nsp)-specific sera were generated, and obtained from Leiden Univ, the Netherlands, for detection of early viral nonstructural proteins to aid in troubleshooting. An equine arteritis virus (EAV) infectious clone was provided by Leiden Univ as a positive control.
SD and ND showed that field strains adapted to grow to high titers in MARC-145 cells were markedly reduced for growth in macrophages. Sequencing of MARC-145-adapted and nonadapted strains is underway to identify attenuation-related mutations. ND is using EAV as a surrogate to develop assays to compare replicative properties of more- and less-virulent PRRSV strains. Advancements were made to quantify transcript accumulation, transcript stability, replicase subunit accumulation, and replicase subunit stability.
KA, with Guelph University, showed that nucleocapsid (N)-eGFP fusion protein localizes to the nucleolus. Individual functional domains were also identified for a cryptic nuclear localization signal (NLS), NLS-1, at amino acid 10, a legitimate NLS, NLS-2, at amino acid 41, a cytoplasmic localization domain adjacent to NLS-2, and a potential nuclear export signal (NES) at amino acid 106. The results suggest that intracellular trafficking of N involves nuclear import, export and cytoplasmic retention signal sequences.
KA reported that amino acids 34-53 of N bind the 5-leader of PRRSV. The RNA-binding sequence is conserved and is unique to PRRSV. KA also reported interactions of cellular proteins from nonpermissive BHK-21 and permissive MARC145 cells with the positive strand of the 3-untranslated region (UTR) and ORF 7 RNA. Virus titer was reduced by 98% by a monoclonal antibody against PRRSV binding proteins of 210, 80, 55, and 30 kDa, indicating a possible receptor complex for PRRSV. CD151, a tetraspan family member, was identified by KA as binding PRRSV 3-RNA. It made BHK-21 cells permissive to PRRSV, suggesting that the CD151 is a susceptibility factor for PRRSV infection.
To explore possible mechanisms for the emergence of acute PRRS in 1996-97, the ORF5 gene of eight acute PRRSV isolates from herds in Iowa and North Carolina was sequenced (VA). They shared 87-97% nucleotide sequence identities with North American PRRSV isolates and vaccines. Isolate 98-37120-2 from a non-vaccinated herd in Iowa was very close to Ingelvac PRRS MLV. The other seven isolates were more related to earlier PRRSV isolates and PrimePac vaccine. The data do not fully support the hypothesis that acute PRRS is due to reversion of vaccines to a pathogenic phenotype. IA and VA collaborated on comparison of recent PRRSV field isolates to Ingelvac PRRS MLV vaccine, and its parent strain, VR2332. The results indicated that vaccine-like isolate 98-38803 is a derivative of Ingelvac PRRS MLV and that the isolate is pneumovirulent.
Low virulent isolates of PRRSV and porcine respiratory coronavirus together enhance respiratory disease and lesions (IA). IA and VA also showed that circovirus-2 infection increases the severity of PRRSV-induced interstitial pneumonia and that circovirus-2, but not PRRSV, induces pathology characteristic of postweaning multisystemic wasting syndrome (PMWS).
Objective 2. Mechanisms of transmission that facilitate PRRSV area spread and persistence in the environment were studied by MN under conditions that replicated commercial swine management practices and building design. Airborne transmission occurred across an airspace of 2 meters, but did not occur from within the facility to swine housed 1 meter outside and directly in line with an exhaust fan. Routing exhaust air into a housing unit containing naive pigs also failed to produce infection or seroconversion. Neither Mallard ducks housed inside a facility with infected pigs, nor experimentally infected Mallard and Rouen ducks, seroconverted or become viremic (MN, SD, IA, NE).
Mechanical transmission of PRRSV by mosquitoes and houseflies occurred when they were fed on infected pigs. Virus was retained in the midgut for 6-12 hr. Interruption of feeding, followed by exposure to naive pigs, resulted in transmission and infection in recipient pig. Fomites, including packed snow, also were demonstrated to mediate transmission of PRRSV into biosecure facilities. These findings demonstrate that PRRSV can be disseminated across considerable distances in a disconnected fashion in nonfreezing and freezing weather.
Viral persistence in pigs was studied by IA, NE, SD, MS, and IL. Animals on commercial farms were persistently infected and re-infected, and multiple viral types coexisted on farms (IL). The contribution of viral evolution to persistence in animals was studied by IA and MN. Multiple variants in ORF 5, but not ORFs 1b (replicase) and 7, were present concurrently in every pig. Most nucleotide changes in ORF5 resulted in substitution of amino acid residues. Virus mutation may not play a role in persistence, since overall genetic change was and there was no evidence of antigenic change (IA). PRRSV was detected in the tonsil of 5 of 8 boars that were negative in serum, semen and PBMC for 2-3 weeks (SD), but in 1 boar at 88 days post-inoculation, PRRSV could not be found by VI or PCR in any of 22 tissues, serum or semen.
Objective 3. To determine if multiple PRRSV exposure induced a state of nonresponsiveness in breeding age swine, pigs were infected with field virus following a 6/60 protocol widely used for sow vaccination (NC). 20% of 55 pigs returned to IDEXX ELISA seronegative status following six homologous wild-type virus infections, even though all pigs had initially seroconverted. Neutralizing antibody was detected in all animals throughout the study. 12 months after initial exposure, animals were infected with homolous (SD 23983) or heterologous (Powell) PRRSV. PRRSV was detected in some animals by PCR, suggesting that they were not fully protected. Only modest anamnestic ELISA and SN antibody responses were observed after homologous virus challenge, but marked increases occurred after heterologous challenge.
Several lines of evidence suggest that the immune response to PRRSV is weak. MN and IL showed that the cytokine IL-12 enhanced interferon (IFN) gamma responses of lymphocytes to PRRSV, and the mucosal adjuvant, cholera toxin, induced a robust antibody response to GP5. Further studies by IL and NE showed that co-delivery of IL-12 or porcine IFN alpha cDNA with PRRS MLV vaccine increased the primary PRRS-virus specific IFN gamma T cell response. Addition of IL-12 to a killed vaccine (PRRomise, Intervet) modestly increased IFN gamma response and antibody titers (IA). IL also showed that most pigs develop low or no titers of virus-neutralizing antibodies. A positive correlation was observed between the frequency of PRRS virus-specific IFN gamma secreting cells and the number of live-born pigs in immune-challenged sows (IL). In vaccinated sows at a commercial facility, only animals with a frequency of >150 PRRS virus-specific IFN gamma secreting cells per million PBMC did not abort following a natural outbreak of PRRS. The significance of the IFN gamma response is not fully understood, since there is a large variation in the response of individual pigs (IL and MN).
To evaluate the protective immune effect of structural proteins, pigs were immunized with Powell strain recombinant ORFs 2-5 (outer membrane proteins) and matrix protein (M) expressed in insect cells (NC). Vaccination with any of the recombinant proteins reduced levels of infectious virus, prevented fever, and prevented behavioral depression. Strong DTH reactions occurred against ORFs 4 and 5, the immunogens that reduced viral load most substantially. NE and IA identified an immunodominant, nonneutralizing epitope in GP5, and a different, neutralizing but non-immunodominant epitope that highly conserved among isolates. The nonneutralizing epitope may act as a decoy, eliciting most of the antibodies directed to GP5 and delaying the induction of neutralizing antibody. NE, IL, and SD collaborated on the construction and immunogenicity evaluation of Mycobacterium bovis BCG expressing GP5 and M proteins. Pigs developed specific immune response against the viral proteins, and 3 of 5 animals developed neutralizing antibodies. An IFN gamma response was not developed against the viral proteins. Following virulent challenge, immunized pigs showed shortened fever, lower viremia, and reduced virus in bronchial lymph nodes, consistent with protection.
KA, with Thomas Jefferson University, identified putative genetic suppressor elements (GSEs) in the PRRSV genome that may render cells permanently resistant to virus infection. Four sequences were identified; one encodes an antisense RNA matching the 5 end of ORF1b, and the others encode sense RNA matching sequences in nsp 11, ORF 2 and ORF 6.
Objective 4. SD and MN developed a blocking N ELISA. Sensitivity was 97.3% and specificity was 100%. CV was 4.12%. 147 of 4142 serum samples from PRRSV negative farms were unexpectedly positive by the IDEXX ELISA (SD, MN, IA). 100% of the 147 samples were negative by IFA and 97% were negative by blocking ELISA. Thus, blocking ELISA is an alternative to IFA for determining the PRRSV serostatus of individual animals. IA, SD, MN, KA, and NE determined that the rate of suspected-false- IDEXX-positive reactors is 0.5-2%. As IDEXX recently introduced a new version of PRRS ELISA, its diagnostic performance needs to be evaluated.
Serodiagnosis of European-genotype PRRSV in the US was improved by SD. European-like PRRSV isolates were grown for improved IFA and virus neutralization assays. 60 monoclonal antibodies recognized Lelystad virus-like isolates. One monoclonal antibody against N protein recognized the US European-genotype PRRSV isolates but not the Lelystad virus or other European isolates. Phylogenetic analysis of the structural protein sequences indicated that these isolates are typical European genotypes. Some of the unique characteristics of these European-like isolates can have a significant impact on diagnostic approaches to detecting PRRSV in US herds.
Current PRRS diagnostic tests were evaluated for detecting PRRSV infection in fetuses. PCR showed the best performance. Submission of whole litters and more than one litter is critical for accurate diagnosis as distribution of the virus among fetuses is not uniform (SD).
Quantitative, real-time PCR assays were developed for the detection of PRRSV by SD and MN. Probes that differentiate US and European PRRSV field and vaccine strains were made (SD). A heteroduplex mobility assay for rapid differentiation of vaccine-like viruses from field viruses was developed and compared to sequencing (VA, IA). The assay was shown to effectively detect and differentiate field isolates from vaccine strains. Isolates with 2% sequence divergence from the vaccine were all determined to be wild-type.
A porcine respiratory disease complex (PRDC) microarray is being developed to simultaneously diagnose PRRSV, influenza A, circovirus 2, Pasteurella multocida, Mycoplasma hyopneumoniae, Streptococcus suis, Actinobacillus pleuropneumoniae, Bordetella bronchiseptica, Haemophilus parasuis, Leptospira species, and porcine parvovirus (MN).
Impact Statements:
- An infectious clone will provide both a direct method for studying the role of viral genes in the pathogenesis of PRRSV as well as the development of a genetically-engineered marker vaccine.
- Transmission studies that confirm previous observations of limited airborne and avian spread of PRRSV, combined with demonstrations of fomite and insect-born spread of virus to biosecure facilities, address producer concerns about the inability to effectively prevent PRRSV introduction to noninfected herds by showing that current practices may not be sufficient.
- Studies of genetic variation during persistent infection of animals shows that PRRSV persistence is not dependent on immune evasion or neutralization escape. It increases the likelihood that persistence in animals is due, at least in part, to a weak immune response.
- Antibody and T lymphocyte responses are both important for an effective immune response to PRRSV. More specific immune assays that measure functionally relevant responses to defined antigens are expected to identify improved correlates of protective immunity to acute and persistent infection. These findings will facilitate development of more efficacious vaccines.
- The blocking ELISA to resolve false-positive serology directly addresses a critical producer concern. Reagent development for serological diagnosis of European-like field cases further enhances the accuracy and reliability of PRRS serology.
- Direct, rapid and sensitive PCR tests for infection status that differentiate US and European genotypes and distinguish between field and vaccine isolates will provide producers and veterinarians will greatly improved tools to assess the infection or protection status of herds and individual breeding animals.
- Plans for the group are to develop a public, international workshop on PRRS advances, publish literature reviews built around the four programmatic objectives, and to investigate development of an international scientific training program for PRRS research.
Last Modified:
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Date of Annual Report: 02/03/2004
Report Information:
Participants:
URL: Copy of participant list
Brief Summary of Minutes of Annual Meeting:
URL: Copy of minutes
Accomplishments:
1. Define molecular and cellular mechanisms of pathogenesis of respiratory and reproductive syndromes caused by PRRSV.Infectious clones of PRRSV were produced by SD in collaboration with Dr. Dongwan Yoo at Guelph University, Ontario, Canada and also shared with KA. A full-length clone of SD23983 containing a consensus 5end based on Genbank sequences of North American PRRSV strains is currently being tested. The infectious clone of VR2332 reported by Neilsen et al. was generously provided to SD and MO by MN. At MO, two rescue attempts with the VR-2332 clone both resulted in successful recovery of fully infectious virus with cytopathic effect and positive fluorescent-antibody staining indistinguishable from that of other PRRSV isolates. An infectious clone of PRRSV was constructed by NE from a highly abortifacient strain.
New 184 field isolates at MN have regions of radical dissimilarity all through the genome. SD and MN characterized European-like PRRSV isolates identified in the U.S. SD and KA focused on heterogeneity in the Nsp2 gene and a preliminary evaluation of phylogeny of these isolates. The ORF 1 of a European-like PRRSV isolate, SD 01-08, is organized the same as LV. The principal difference was a 17 aa deletion in Nsp2 of SD 01-08 that is not consistently observed. Evolutionary relationships among the European, North American and European-like isolates showed evidence for nucleotide diversity, including several deletions in the Nsp2 region. The European-like PRRSV isolates in the U.S. appear to be from a single Lelystad-like isolate.
KA and SD determined that the ratio of 2b to N in virion preparations was estimated to be approximately 20 N protein molecules for each 2b molecule. Thus 2b is a minor component of the PRRSV virion. KA and Guelph University determined that a single nuclear localization signal (NLS) is involved in the transport of N from the cytoplasm and into nucleus.
VA and IA collaborated on molecular analyses and biological characterizations of PRRSV field reisolates of vaccine and determined that the vaccine-like isolate 98-38803 induces microscopic pneumonia lesions similar in type to, but different in severity and time of onset from, those observed with virulent strains VR2385 and the parent strain of the vaccine.
Quasispecies diversity was analyzed in PRRSV in tonsils from 2 chronically infected farms in IL. All animals in the study harbored multiple, distinct PRRSV variants at both the nucleic acid and amino acid levels. The results indicate that PRRSV exists during natural infection as quasispecies distributions of related genotypes, but that positive natural selection for immune evasiveness in ORF5 does not appear to contribute to maintaining this diversity.
NE and IA conducted studies that indicated genetic variation in susceptibility to PRRSV. Future research with tissues collected will determine which genes are expressed differently in pigs with resistant and susceptible responses to PRRSV.
2. Determine the mechanism(s) and consequences of viral persistence.
NC developed and tested a serologic monitoring method to estimate the point-in-time portion of animals that are resistant to PRRSV, the portion that are susceptible, and the portion of animals that are infective. SD and MN examined the role of lymphoid and non-lymphoid tissue in acute and persistent infections of PRRSV. PRRSV disseminated to most tissues and lymph nodes in 2 d and was detected out to 70 dpi in various tissues. Lymphoid tissues play a prominent role as primary and persistent sites for PRRS viral replication.
MN detected PRRSV in flies in baited jug traps up to 1.66 km from an experimentally infected herd. Hence, transmission and persisttence of PRRSV in swine herds appears to involve flies and mosquitoes. Transport vehicles were also shown to effectively transmit PRRSV, but washing, disinfecting and drying eliminated PRRSV. Drying appeared to be an important component of a transport vehicle sanitation program for PRRSV biosecurity. Aerosolized PRRSV was transported 150 meters in a straight tube model and was infectious to susceptible pigs (MN). Attempts to infect mallard ducks with PRRSV via direct inoculation were not successful.
3. To characterize the different components of the immune response during acute and persistent infection and its implications in diagnostics and pathogenesis.
KA determined that the thymus is the principal site of virus replication in the fetus. IFN gamma and TNF were induced in lung and lymph nodes. IL and USDA-BARC reported an IFN gamma response to PRRSV after 5 weeks. A significant up-regulation of TNF indicated the involvement of innate immunity to PRRSV starting early in the infection, yet there was limited up-regulation of IFN alpha. Co-administration of IFN alpha caused faster returns to pre-vaccination levels for IL-6, IL-8 and IL-10 after 4 weeks. Co-administration of IFN alpha enhanced the IFN gamma response to vaccine. NE and IA produced chimeric mouse X pig anti-PRRSV antibodies that reacted with GP5 in ELISA and Western blot.
MN, NC, and IA collaborated to examine humoral immune responses to structural and nonstructural (nsp) proteins. PRRSV nsp 1, 2, and 4 were cloned. Direct ELISA to nsp 1 revealed that peak levels of antibody exceed anti-PRRSV nucleocapsid (N), and the levels were maintained for at least 120 days. Antibodies to nsp 4 appeared at low levels for a short time.
MN and IA collaborated to report that PRRSV-specific T cell responses were transient and varied substantially among animals. Viral loads decreased 1,000-fold in persistent infections, with tonsil being the primary site of persistence. A weak CMI response appears to contribute to prolonged PRRSV infection and suggests that PRRSV suppresses T cell recognition of infected macrophages. IL monitored the kinetics of the PRRSV-specific IFN gamma response in PBMC for 9 months. A weak IFN gamma response within a few weeks after vaccination appeared to wane at 10 weeks, then rebounded and, with fluctuation, increased gradually in intensity after a period of months without a booster immunization.
IL and NE exposed replacement gilts to a wild-type virus or a heterologous commercial vaccine. A higher IFN gamma response was associated with a lower proportion of stillborn pigs per litter. There was no relationship between humoral or cellular immune response and the recovery of PRRSV from the tonsil. ELISA antibodies were not predictive of any production variables. The use of a heterologous killed PRRS virus vaccine, following exposure to live virus, significantly boosted the cell-mediated immune response.
The role of dendritic cells (DC) in PRRSV pathogenesis and persistence was studied by SD and MN. PRRSV grew in pig DC and reduced expression of MHC class I, CD14, CD11b/c, but not MHC II. MN found no change of expression MHC class I, class II or costsimulatory molecules CD80/86. However, infected DC were impaired in antigen presentation. PRRSV appears to suppress DC function.
Clinical signs and cellular and humoral immune responses were examined at MN in gilts challenged with heterologous or homologous strains 120 days after primary infection. No clinical signs were observed following homologous challenge. Mild clinical signs of fever, anorexia, and depression were observed in all heterologous challenge groups. Viremia was detected in all previously exposed groups challenged with all heterologous strains only on day 3 pi, and not at days 7 and 14 pi. Following homologous challenge, a vigorous CMI response and a weak humoral response were observed. Following heterologous challenge, weak or no CMI responses and vigorous humoral responses were observed.
Development of recombinant PRRSV vaccines using a herpes virus-based amplicon vector system containing the ORF 5 gene of PRRSV 23983 was performed by SD in collaboration with the U of Rochester. ORF 5 protein was detected by western blot using a PRRSV immune serum.
IL established a correlation between the two allelic forms of IL-12R?2 and the intensity of the interferon IFN-? response of swine to immunization with a PRRS MLV vaccine. The IL-12R?2 allele represents the first genetic marker in pigs associated with variation in the degree of cell-mediated immune response to a pig virus.
MO showed that viral growth on macrophages in the presence of interferon alpha was reduced in a manner dependent of viral isolate. PRRSV also suppressed IFN production in response to infection with TGEV, PRCV, polyI:C and VSV, all known IFN inducers. These results demonstrate that PRRSV field isolates differ both in IFN alpha sensitivity and induction. Furthermore, PRRSV field isolates differ in suppression of IFN induction by other viruses and double-stranded RNA.
IA identified anti-PRRSV antibody responses that were enhancing, neutralizing and neither. Identification of the epitopes responsible for enhancement or neutralization may provide the basis for developing efficacious second-generation vaccines. Serological characterization of auto-anti-idiotypic antibodies indicated that the Aab-2s recognized a site within or near the antigen-combining sites of the anti-GP5 antibodies. Aab-2 antibodies may play a role in immunity to PRRSV infection.
4. Develop improved methods for the diagnosis of PRRSV clinical disease and detection of virus and/or antibodies in PRRSV.
Real-time rapid PCR detection of PRRSV and vaccine in serum and semen was evaluated at KA and MO. North American and European ORF7 were amplified using a single set of PCR primers and distinguished by distinct melting peaks. Thus, detection of PRRSV RNA in semen is possible. At MO Lelystad strain and US/European PRRSV field isolates were successfully amplified. Multiplex assays incorporating both PRRSV genotypes and SIV also were developed. SD and NE, with Tetracore, Inc., validated a real-time PCR with primers specific for U.S. or European-like PRRSV. 427 semen and serum samples showed 95% agreement with nested RT-PCR. IA developed a multiplex RT-PCR assay for PRRSV, SIV and PCV2 in nasal swabs and lung lavage.
VA and IA developed a heteroduplex mobility assay (HMA) for quickly identifying PRRSV isolates similar to vaccines. The HMA results also correlated well with the results of phylogenetic analyses and indicate that HMA could be used as a rapid and efficient method for large-scale screening of potential vaccine-like PRRSV field isolates.
SD and MN validated a monoclonal antibody-based blocking ELISA using sera from 686 individual animals of known PRRSV status. Agreement with IDEXX ELISA and IFA for detection of seroconversion was demonstrated. IA also showed that blocking ELISA against nucleocapsid is sensitive and specific. NE, MS and SD determined that antibodies could be raised to PRRSV in ducks and rabbits. An experimental infection of Rouen and Mallard ducks showed no evidence of infection.
NC and SD collaborated to evaluate antibody responses in mature swine following repeated exposure to a single isolate of PRRSv by IDEXX 2XR commercial ELISA. All animals developed solidly positive antibody responses initially on HerdChek ELISA, but not on the HerdChek2XR. ELISA antibody levels dropped substantially 4 months after initial seroconversion, even in the face of repeated injections with virulent SD 28983 PRRSV. All animals remained FFN antibody positive at the end of the study. Challenge of the multiply exposed seronegative animals with a heterologous strain of PRRSV resulted in marked anamnestic ELISA and SN antibody responses.
IA, SD, and KA investigated PRRS ELISA false positive reactors. Ninety seven of 12,000 animals in PRRS-negative swine herds tested as false-positive by IDEXX ELISA. They were evaluated on 3 alternate ELISA-based serologic assays (developed at ISU, SDSU, and KSU) and were negative on at least one test. No test was 100% reliable. Western blotting did not reveal the cause of false positives.
Impact Statements:
- Full-length infectious clones of virulent and attenuated PRRSV strains of North American and European genotypes will be powerful tools for elucidation of the genetic similarities and differences in PRRSV isolates that control phenotypic variation in pathogenesis and immune sensitivity as well as assist the development of a genetically-engineered marker vaccine of PRRSV.
- Understanding the genetic basis for host susceptibility to disease, and the role of concurrent infections in pathogenesis will help to develop disease resistant pigs based on genetically desirable traits.
- Information of the various routes of PRRSV transmission is essential to protect commercial swine operation from becoming infected.
- Knowing that lymphoid tissues play a prominent role as both primary and persistent sites for PRRS viral replication will improve studies of disease pathogenesis and immune protection.
- PRRSV Nsp2 may be useful as a marker for diversity within the group of European-like and North American PRRSV isolates.
- Understanding the mechanisms that regulate the development of anti-viral protective immunity will lead to the development of more effective vaccines and adjuvants, and assist in developing better methods to monitor immunity.
- Knowledge of viral mechanisms of immunosuppression, especially suppression of the innate immune response, will help elucidate improve methods of PRRSV control through vaccination.
- Studies with dendritic cells will provide insight into the mechanisms for viral persistence and immune modulation associated with PRRSV, while new vaccine strategies may eventually offer improved methods for the control of PRRS.
- Application of real-time, quantitative PCR assay has broad potential for commercial use in boar studs, diagnostic laboratories and other facilities for monitoring PRRSV infection and biosecurity for the detection of U.S and Lelystad or European-like PRRSV.
- The blocking ELISA offers an alternative to the IFA for follow-up of suspect PRRS serological results.
- The use of cell culture adapted European-like PRRSV isolates of U.S. origin in routine diagnostic IFA and virus neutralization assays allows for improved serological monitoring without the restrictions associated with imported European isolates, such as LV.
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