Diagnosis of PRRS VirusK-J Yoon, J Christopher-Hennings, EA Nelson

• Due to the similarity of clinical signs of PRRS virus infection and those caused by other viral and bacterialpathogens, laboratory tests are required to definitively diagnose PRRS.

• Several kinds of serologic tests are available include enzyme-linked immunosorbent assay (ELISA), serumvirus neutralization (SVN), and indirect fluorescent antibody (IFA). These tests only indicate that a pig hasbeen exposed to the virus naturally or through vaccination but cannot tell if the pig remains infected.

• Additional tests are available to actually determine the presence of the virus. These tests includeimmunohistochemistry staining (IHC), fluorescent antibody staining (FA), polymerase chain reaction (PCR),and virus isolation (VI). While a positive result on these tests indicates the virus is present in the sample, anegative test does not necessarily indicate that the pig is free of the virus. Correct sample selection, samplehandling, and the sensitivity of the test all interact to provide a reliable result.

• No serologic test can differentiate whether a pig has been infected with a field strain of PRRS virus or has onlybeen vaccinated.

• Genomic sequencing of the virus can predict with some accuracy the relatedness between two strains of PRRSvirus as well as how closely related they are to vaccine strains. Genomic sequencing will not predict thelikelihood of a vaccine to successfully prevent the disease.

• Restriction fragment length polymorphism (RFLP or “cut pattern”) has limited value as a PRRS virusdiagnostic test.

• Pigs born to seropositive dams will remain positive on serologic tests until 3 to 16 weeks of age. The exacttiming will vary according to the level of positivity in the dam and what serologic test is being used.

• Since antibodies do not persist for the lifetime of an animal, it is generally recommended that young pigs, ratherthan breeding stock, be tested to determine a herd’s PRRS virus infection status.

57

Diagnosis of PRRS VirusK-J Yoon, J Christopher-Hennings, EA Nelson

Introduction

A tentative diagnosis of PRRS virus infection issuggested by clinical signs; reproductive problems inbreeding stock or respiratory disease in pigs of anyage. Reproductive problems associated with PRRSinclude poor conception rates, late-term abortions,and an increase in the rate of stillborn pigs,mummified fetuses, and weak, non-viable piglets.Infection with PRRS virus often does not induceunique gross or microscopic lesions, althoughinterstitial pneumonia is a common finding in therespiratory form. In addition, the gross lesionscaused by PRRS virus infection may resemble or beobscured by lesions caused by other infectiousagents.

Due to the similarity of clinical signs of PRRS virusinfection to those caused by other viral and bacterialpathogens and the lack of PRRS virus-specificlesions, differential tests are required for a definitivediagnosis. The differential diagnosis includesinfection with porcine parvovirus, pseudorabies virus,hemagglutinating encephalomyelitis virus, porcinecircovirus type 2, porcine enterovirus, swineinfluenza virus, classical swine fever (hog cholera)virus, porcine cytomegalovirus, and leptospirosis(Allan and Ellis, 2000; Keffaber, 1989; Halbur et al.,1993, 1995a; Mengeling et al., 1993; Paton et al.,1992b; Yoon et al., 1996a). Therefore, when theclinical history and pathology is suggestive of PRRS,detection of viral antigens, viral genomic material, orisolation of virus from clinical specimens must beused to confirm the tentative diagnosis.Alternatively, documentation of rising serumantibodies in a time frame compatible with theclinical episode may support the diagnosis. Thischapter describes laboratory procedures for thediagnosis of PRRS virus and approaches fordiagnostic investigation to confirm PRRS virusinfection in a herd.

General Guidelines for PRRS Diagnosis

Diagnosis is based on obtaining evidence of PRRSvirus infection in animals suspected of harboring theinfection. Such evidence may be obtained byisolating PRRS virus or detecting PRRS viralantigens or nucleic acid in the animals. In clinicallyaffected animals, demonstration of suggestivepathological lesions supports the diagnosis of PRRSin conjunction with laboratory testing. It should be

kept in mind that PRRS virus or PRRS viral RNAcan be detected in clinically normal animalsvaccinated for PRRS or animals persistently infected.Timing after vaccination or history of previousexposure should be taken into consideration whenconducting a diagnostic investigation for PRRS.

Successful isolation and/or detection of viruses inclinical materials are highly dependent on propercollection and handling of specimens. In general,specimens intended for virus assays should becollected as early as possible in the course of thedisease, i.e., within the first 7 to 10 days after theonset of illness. Samples collected during the acutephase of viral infection usually contain adequateamounts of virus for detection in available assays.Samples collected later in the course of infectionusually require more laboratory time and often yieldpoor or negative results. It is important to choose notonly the most appropriate specimen, but also tocollect an adequate amount of specimen forvirological testing. Insufficient amounts of sampleare a potential cause of inconclusive diagnosis orfalse-negative result.

For best results in isolation and detection of viruses,clinical specimens should be aseptically collected,kept fresh, and transported immediately to thelaboratory. If delays are unavoidable or anydetrimental affects on virus in samples are anticipatedduring transport, samples should be refrigerated at40º F (4º C) for no more than 2 days. For longerstorage periods, freeze samples at minus (–)70º C, butNEVER at minus (–)20º C. Self-defrosting freezersin conventional refrigerators are not appropriate forstorage. Ideally, frozen samples should be submittedon dry ice, but commercial refrigerant packs can beused if necessary.

Demonstration of seroconversion or increasing titersof PRRS virus-specific antibody in a group ofaffected animals can also be used for diagnosis ofPRRS virus infection. One must exercise cautionwhen interpreting serology in herds already known tobe infected or vaccinated. This is due to the fact thatnone of the available serologic tests can differentiatepositive results due to infection from positive resultsdue to vaccination. Additionally, serology can not berelied upon to determine how long a pig has beeninfected. Serologic testing coupled with an

58

epidemiological approach, such as case-control orlongitudinal studies, strengthens the use of serologyfor PRRS diagnosis. When herd monitoring isdesired, utilizing other tests such as polymerase chainreaction (PCR) or virus isolation (VI) in combinationwith serology will give the producer and veterinariana clearer picture of virus transmission patterns.

Assays for Detection of PRRS Virus

Isolation of live virusPRRS virus is known to replicate in only two types ofcells: porcine alveolar macrophages (PAMs) andcertain African monkey kidney cell lines (Bautista etal., 1993b; Dea et al., 1992; Paton et al., 1992a,1992b; Wensvoort et al., 1991; Yoon et al., 1992b;Zeman et al., 1993). However, all PRRS virusisolates do not replicate in both cell types. Thissuggests that at least two cell types should be usedfor virus isolation whenever possible. Use of PAMsmay be required when attempts to isolate European-like PRRS virus are made (Dewey et al 2000;Wensvoort et al., 1991; M. Murtaugh personalcommunication).

PRRS virus has been isolated from a variety ofclinical specimens (Done et al., 1996; Goyal 1993;Paton et al., 1992b; Rossow, 1998), including serum,plasma, peripheral blood mononuclear cells (i.e.,buffy coat layer), bone marrow, tonsil, lungs, lymphnodes, thymus, spleen, heart, brain, liver, testis,epididymus, ductus deferens, bulbourethral gland,penile tissue, oropharyngeal scraping, nasal turbinate,nasal swabs, placenta, saliva, urine, feces, and semen(Baron et al., 1992; Benfield et al., 1994a, 1994b;Christianson et al., 1992, 1993; Christopher-Hennings et al., 1995b, 1998, 2001; Dea et al., 1992;Done et al., 1992; Goyal and Collins, 1992; Horter etal., 2002; Joo, 1993; Keffaber et al., 1992; Mendez-Trigo, 1993; Ohlinger et al., 1992; Paton et al.,1992a, 1992b; Rossow et al., 1994, 1999; Rowland etal., 1996; Swenson et al., 1994; Thacker, 1992; VanAlstine et al., 1993a, 1993b; Wills et al., 1997a,1997b; Yoon et al., 1993). Among all samplesdescribed above, fluid collected from deep with thelungs (bronchioalveolar fluid) and serum areconsidered by some as the preferred specimens forvirus isolation when an acute PRRS outbreak occurs,regardless of the age of the affected animals. PRRSvirus is more stable in serum than in tissue(Mengeling et al., 1995; Rossow, 1998; Van Alstineet al., 1993a). In older animals, viremia is of shortduration and PRRS virus may be found in tissueslonger than in serum (Christopher-Hennings et. al.,2001). If tissues are submitted, they should include

lung, tonsil, and lymph nodes (Joo, 1993). Tissuemust be fresh if virus isolation is to be successful,regardless of which tissues are submitted fordiagnostic investigation.

Sample selection may also depend on the stage ofinfection (i.e., acute, convalescent, or persistent).Serum, lung, and bronchioalveolar lavage fluid aresamples of choice for isolation of PRRS virus inacutely infected animals (Joo, 1993; Mengeling et al.,1995, 1996a, 1996b). For virus isolation frompersistently infected animals, tonsil, orophargynealscraping, and bronchioalveolar lavage fluid are bettersamples than serum and lung (Christopher-Henningset al., 2001; Horter et al., 2002; Mengeling et al.,1995, 1996a, 1996b; Wills et al., 1997a). In cases oflate-term abortion and early farrowing, samplesshould be collected from weakborn, pre-suckle pigs,rather than mummies, aborted, or stillborn pigs (Doneet al., 1992; Joo 1993; Van Alstine et al., 1993a;Zeman et al., 1993). Within affected litters,weakborn pigs are the most likely to be viremic, butthe presence of high levels of maternal antibody toPRRS virus may hinder attempts at virus isolation.

The survivability of PRRS virus in diagnosticspecimens exposed to different environmentaltemperatures has been evaluated in clinicalspecimens (lung, spleen, thymus, and serum). Thecurrent recommendation is that tissues and clinicalspecimens for virus isolation be kept at 4º C (40º F)or lower following collection and during shipment toa diagnostic laboratory in order to enhance thelikelihood of isolating virus (Van Alstine et al.,1993a, 1993b). If long-term storage of samples isnecessary, specimens should be kept frozen at minus(–)70º C without repeated cycles of freeze-thawing.

Detection of virus or antigen (live ordead)The frozen tissue section fluorescent antibody test(FA) and immunohistochemistry (IHC) test may beused for detecting PRRS virus antigen in tissues. Thedirect FA test on frozen tissue sections is inexpensiveand rapid. The test is specific (has few false positiveresults), but is not always very sensitive (tends toresult in a few false negative results). Sample qualitygreatly affects FA test results. Tissue should becollected from recently dead or euthanized pigs andpromptly refrigerated or frozen. In contrast, IHC isuseful for detecting virus in formalin-fixed tissues.IHC is more sensitive than direct FA examination offrozen tissues, but takes more time and is moreexpensive than the FA test. A definitive diagnosiscan be accomplished by detection of microscopic

59

lesions characteristic of PRRS virus in conjunctionwith IHC or FA tests.

For direct FA examination, fresh or frozen tissuesshould be submitted. For IHC examination, tissuesshould be fixed in 10 percent neutral bufferedformalin. Preferred tissues for these tests are heart,kidney, lung, lymph nodes, spleen, thymus, andtonsil. PRRS viral antigens may also be detected inthe adrenal gland, intestine, liver, and occasionally inthe brain (Halbur et al., 1996; Rossow et al., 1999).When performing antigen detection tests, such as FAand IHC, laboratories must choose whether to test forthe US or European PRRS virus. Veterinariansshould familiarize themselves with their locallaboratory to understand which test should be run.

Genetic-based testing for PRRS virusPolymerase chain reaction (PCR)-based tests havebeen developed for detecting PRRS virus RNA inclinical specimens. Since the virus does not need tobe isolated in cell culture to detect the viral RNA,PCR can be performed in a shorter amount of timethan virus isolation. As a general principle, PCR-based assays are believed to be highly sensitive andhighly specific (Benson et al., 2002; Horter et al2002).

Several types of PCR-based assays have beendeveloped, most being designed to detect regions ofthe virus called ORF7, ORF6 or ORF1b and can berun directly on diagnostic specimens (Chen andPlagemann 1995; Christopher-Hennings et al., 1995a;Cook and Spatz, 1998; Egli et al., 2001; Gilbert et al.,1997; Legeay et al., 1997; Mardassi et al., 1994;Oleksiewicz et al., 1998, Shin et al., 1997;Spagnuolo-Weaver et al., 1998; Suarez et al., 1994;Van Wonsel et al., 1994). Some PCR assays use a“nested” procedure (RT-nPCR) for added sensitivity.More recently, automated fluorogenic PCR-basedtests, such as the TaqMan™ PCR (Egli et al., 2001;Spagnuolo-Weaver et al., 2000) or “MolecularBeacon” PCR (Carlson et al., 2002) have beendeveloped for detecting PRRS virus in clinicalspecimens. These PCRs are believed to improve thereliability and consistency of conventional PCR testsfor PRRS virus detection.

Historically, the primary diagnostic application ofPRRS RT-PCR has been for the detection of virus inboar semen (Christopher-Hennings et al., 1995a,1995b, 1996; Shin et al., 1997). PCR is particularlyuseful for the detection of virus in samples likesemen or feces because these samples are difficult toanalyze by traditional methods. It has also beenfound useful for detection of PRRS virus in fetal

tissues and thoracic fluids (Benson et al., 2002). Theuse of PCR assays has become more common bothfor the diagnosis of PRRS and to aid in herdmonitoring, i.e., screening of replacement animals,detection of persistently infected animals, and testand removal programs (Bierk et al., 2001; Dee et al.,2001a; Horter et al., 2002; Kleiboeker et al., 2002).The performance of PCR testing among differentlaboratories may vary depending upon samplecondition, sample processing, laboratory technique,and the skills and experience of the technicianperforming the assay. Therefore, it is important forlaboratories performing PCR to validate their assaysand provide this information to producers andveterinarians using their specific PCR test.

Assays for Detection of Serum Antibodies(Serology)

The indirect fluorescent antibody (IFA) test, serumvirus neutralization (SVN) test, immunoperoxidasemonolayer assay (IPMA), and enzyme-linkedimmunosorbent assay (ELISA) have all been used forthe detection of antibodies specific for PRRS virus.The IFA, SVN, and ELISA are currently available inmost North American veterinary diagnosticlaboratories, while IPMA has been extensively usedin Europe.

The IFA is thought to have high specificity (99.5%)but unknown sensitivity for individual animals (Yoonet al., 1992a). An advantage of the IFA testcompared to ELISA is that the magnitude of theantibody titer can be determined. An antibody titerof 16 or 20, depending upon the initial serum dilutionfor the test, is considered positive. The IFA reliablydetects specific antibodies for 2 to 3 months afterinfection (Frey et al., 1992; Yoon et al., 1995b). Testresults or endpoint antibody titers will varydepending on the degree to which the PRRS virusstrain used in the assay differs from the isolate thatinfected the pig (Bautista et al., 1993a).

The IPMA is also considered to be a highly specificand sensitive test (Wensvoort et al., 1991). In onecomparative study, the sensitivity of IPMA wasbetter than that of a commercial ELISA (Drew,1995). Antibodies to PRRS virus are usuallydetected by IPMA between 7 and 15 days afterinfection (Ohlinger et al., 1992; Wensvoort et al.,1991). Like the IFA, IPMA also reliably detectsspecific antibodies for 2 to 3 months after infection(Frey et al., 1992; Ohlinger et al., 1992; Yoon et al.,1995a). The relatedness of the virus strain used inthe assay and the virus strain infecting the pig will

60

likely affect the performance of the IPMA test(Wensvoort et al., 1992).

The ELISA is also reported to be sensitive andspecific (Albina et al., 1992, Edwards et al., 1994;Nodelijk et al., 1996; O’Connor et al., 2002;Takikawa et al., 1996). One disadvantage of theELISA format reported during the developmentalstage was an unacceptable number of false positiveresults (Edwards et al., 1994; Paton et al., 1992b).Several ELISA formats have been described: anindirect ELISA using a sample to positive (S/P) ratiosystem (Yoon et al., 1995a), an indirect ELISA usingdirect OD values (Albina et al., 1992; Cho et al.,1996; Takikawa et al., 1996), and a blocking ELISA(Ferrin et al., 2002; Houben et al., 1995a; Sorensen etal., 1997; Zhou et al 2001). In the commercialELISA kit (HerdChek® PRRS ELISA, IDEXXLaboratories Inc., Westbrook, Maine), a S/P ratio≥0.4 is considered positive. Using the S/P ratio of0.4 as a cut off, PRRS virus-specific antibody isdetected in young pigs between 10 to 14 days postinoculation under experimental conditions and peaksat 2 to 3 months (Yoon et al., 1995a). The specificityof the HerdChek® PRRS ELISA has been estimatedto be between 99.3 and 99.5 percent (O’Connor et al.,2002; Nodelijk et al., 1996). Recently a new versionof the assay (HerdChek® 2XR) has been madeavailable to veterinary diagnostic laboratories andpractitioners. No published information on itsperformance or comparison with the previous assay isavailable at present. Uniformity in the manufacturingof the kit and a high degree of automation inperforming the test in the diagnostic laboratory resultin less variation in the results from the commercialELISA compared to other assays. Other advantagesof the commercial ELISA include: detection ofantibody against North American and EuropeanPRRS virus strains, fast turnaround time, andlicensure by USDA and AgCanada.

The SVN test is also considered a specific test, butprevious studies have suggested that SVN is lesssensitive than IFA and ELISA (Benfield et al.,1992b; Morrison et al., 1992). The low sensitivity ofthe test is primarily due to the fact that neutralizingantibodies (the type of antibody detected by the SVNtest) against PRRS virus develop as late as 1 to 2months after infection (Frey et al., 1992; Goyal andCollins, 1992; Minehart et al., 1992; Morrison et al.,1992; Nelson et al., 1994; Yoon et al., 1995a).Currently, a titer of ≥4 is considered positive. TheSVN test is best considered a research tool ratherthan a routine diagnostic test because of its laboriousnature. As with IFA and IPMA, test results aregreatly influenced by the degree of relatedness

between the isolate employed in the test and theisolate infecting pigs (Wensvoort et al., 1992; Yoonet al., 1997).

Interpretation of Serologic Results

Antibodies specific for PRRS virus often do notpersist for the lifetime of an animal. In pigs exposedto PRRS virus under experimental conditions, virus-specific antibodies were initially detected by the IgG-IFA, IPMA, ELISA, and the SVN test at 5 to 9, 9 to11, 9 to 13, and 9 to 28 days post inoculation,respectively. PRRS virus-specific IgM antibodies aredetected within 5 days post inoculation and persist 21to 28 days post inoculation (Park et al., 1995).Depending on the assay, antibody levels reach theirpeak values by 30 to 50 (IFA), 35 to 50 (IPMA), 30to 50 (ELISA), and 60 to 90 days post inoculation(SVN) (Yoon et al., 1995a), after which they begin todecline. Yoon et al. (1995a) estimated fromexperimental and field observations that antibodytiters approach undetectable levels by 4 to 5 months(IFA), 4 to ≥10 months (ELISA), 11 to 12 months(IPMA), and 12 months (SVN) post infection. Thesame time frame would be expected in those pigs thathave been vaccinated with MLV vaccine (but havenot been previously exposed to wild PRRS virus).

Several problems or limitations should be taken intoaccount when interpreting PRRS serology. Resultsfrom a single blood sample collected from anindividual pig showing clinical signs consistent withPRRS, is generally insufficient for confirming adiagnosis of PRRS (Van Alstine et al., 1993b).Positive results may or may not mean that PRRSvirus caused clinical disease (Henry, 1994). Thepossible presence of maternal antibody should beconsidered when interpreting serology results.Albina et al. (1994) reported that maternal antibodywas detected in the serum of piglets tested 4 daysafter birth and disappeared by 3 weeks of age.Alternatively, maternal antibody specific for PRRSvirus has been reported to persist as long as 4 to 10weeks of age (Goyal, 1993; Houben et al., 1995b;Nodelijk et al., 1997) and occasionally up to 16weeks of age in pigs nursing immune dams (VanAlstine et al., 1993b).

Since antibodies often do not persist for the lifetimeof an animal and because of the relatively shortduration of IFA and/or ELISA antibodies, it isgenerally recommended that young pigs, rather thanbreeding stock, be tested to determine a herd's PRRSvirus infection status. In single-site, farrow-to-finishswine herds, the seroprevalence of PRRS virus

61

infection is usually considered to be highest in thegrow-finish unit.

Negative PRRS serology on samples at one point intime can have several possible interpretations (AASPSubcommittee on PRRS, 1996):

1. The pigs are not infected with virus.2. The pigs were recently infected with virus but

have not yet had time to seroconvert.3. The pigs were infected with virus some time ago,

but have since become seronegative.4. The result was falsely negative due to poor

sensitivity of the test or a laboratory error.

Therefore, if using single point-in-time samples,PRRS serology must be used in conjunction withvalid population sampling methods and knowledge ofa herd’s history to determine if the herd has beenexposed to PRRS virus. Current serologic tests arebetter suited for determining the status of apopulation, not necessarily the status of individualanimals.

Diagnosis of PRRS virus infection as the cause ofreproductive failure or respiratory disease can beachieved by showing a change in antibody titer (i.e.,rising antibody titer) in paired serum samples.However, a definitive diagnostic evaluation of PRRSwith respect to clinical disease requires thatserological information be interpreted in combinationwith results from virus isolation and/or detection ofantigenic or genomic material (Christianson and Joo,1994; Goyal, 1993). It is important to bear in mindthat the current serologic assays used in thediagnostic setting cannot routinely differentiatevaccine-derived antibodies from field isolate-derivedantibodies.

The occurrence of false positive serologic results onthe commercial ELISA has been a concern fordiagnosticians and practitioners, particularly inexpected-negative herds or groups (Keay et al., 2002;O’Connor et al., 2002; Torremorell et al., 2002).Field observations in "expected-negative" herds havesuggested that false positive animals (“singletonreactors”) occur at a rate of 0.5 to 2 percent. Mostsuspected false positive animals have ELISA S/Pvalues around the 0.4 cut off value, but S/P ratios canoccasionally exceed 1.0 in these animals. At present,no information regarding the occurrence of falsepositives is available on the recently released 2XRversion of the HerdChek® ELISA. Currentrecommendations for evaluating suspected falsepositive animals include repeating the test, testing byother serological methods in conjunction with PCR,

re-sampling the suspected false positive animal andre-testing, or occasionally, sacrificing the animal andconducting a complete diagnostic work-up. A fewdiagnostic laboratories have developed in-houseELISAs for more specific detection of positiveanimals (Ferrin et al., 2002; Zhou et al 2001). Suchtests are available on an experimental basis atpresent.

Differential Testing

Serologic assays cannot routinely differentiateantibodies to field isolates from vaccine-derivedantibodies. However, characterization of virusisolates is possible by several methods. Panels ofmonoclonal antibodies can easily differentiateEuropean isolates from North American isolates andvice versa (Dea et al., 1996; Drew et al., 1995;Nelson et al., 1993, 1996; Yang et al., 1999, 2000).Using this technique, Nelson et al. (1996) could findno evidence of the Lelystad virus (LV) or LV-likePRRS viruses in Midwestern US swine herds afterevaluating 306 field isolates collected before 1995.However, recent reports of “EuroPRRS" virus, i.e.,Lelystad-like virus or European PRRS virus in NorthAmerica (Dewey et al., 2000) suggest thatmonoclonal antibody analysis of isolates may beuseful for differentiation of isolates. Monoclonalantibody analysis can also be used to differentiatecommercial modified-live vaccine virus from fieldisolates (Yang et al 2000).

Molecular biology has also made it possible tocharacterize PRRS virus isolates using PCR, arestriction fragment length polymorphism (RFLP)assay (Wesley et al., 1998a; Umthun et al. 1999), anddirect DNA sequencing (Kapur et al., 1996; Yoon etal 2001). A PCR-based technique has beendeveloped to differentiate North American fromEuropean isolates (Christopher-Hennings et al.,1995a; Egli et al., 2001; Gilbert et al., 1997; Mardassiet al., 1994). Although the investigatorsdemonstrated its usefulness, PCR was not routinelyused for that purpose in North American until therecent report of the presence of European-like PRRSvirus in North America (Dewey et al., 2000).Currently, several diagnostic laboratories conductdifferential PCR on suspect cases as per request or atthe diagnostician’s discretion. More recently, aheteroduplex mobility assay (HMA) has beendeveloped for a rapid identification anddifferentiation of vaccine-like virus from field viruses(Key et al., 2002a) but has only limited availability.

The RFLP assay is a crude technique fordifferentiating one PRRS virus isolate from another.

62

It was developed early on in the genesis of PRRSvirus diagnostic tools but has recently fallen out offavor. The RFLP technique involves virus isolationfollowed by restriction endonuclease digestion.Restriction endonucleases are enzymes that makecuts at specific places in a genomic sequence.Different PRRS viruses differ in their genomicsequences, so fragments of different sizes areproduced. The lengths of these fragments are thenassigned a 3-digit number, e.g., 2-5-2 or 1-4-2. TheRFLP pattern of a virus is a symbolic code. That is,RFLP patterns have no known association withspecific viral characteristics, such as virulence orantigenic relatedness, and in that sense, the resultshave limited usefulness. Also, the RFLP cut patternis representative of only a small percentage of theentire genomic sequence.

In contrast to RFLP, direct genomic sequenceanalysis of PRRS virus detects differences andsimilarities between isolates, enabling investigatorsto differentiate viruses at the genetic level.Sequencing provides investigators with the exact andcomplete sequence of the desired part of the genome.The sequencing is able to show nucleotide mutations,additions, and deletions that RFLP analysis maymiss. Sequence analysis and comparison can be auseful tool for veterinarians or investigators who aremonitoring virus spread through pig flow within aproduction system over time. With sequence analysisit is possible to differentiate vaccine from fieldisolates, analyze changes to the PRRS virus thatoccur over time, and describe the “family tree”among various PRRS virus isolates both within and

between populations of animals (Andreyev et al.,1997; Dee et al., 2001b; Key et al., 2001; Meng,2000; Murtaugh et al., 1995; Nelsen et al., 1999;Pirzadeh et al. 1998; Rowland et al., 1999).

Numerous field-based descriptive studies haverevealed remarkable genetic variability among PRRSviruses (Andreyev et al. 1997; Kapur et al., 1996;Key et al., 2001b; Meng 2000), suggesting that PRRSvirus is rapidly evolving over time. However,experiments examining PRRS virus mutation havenot explained all of the observed variability seenamong field isolates. Under experimental conditions,the divergence between the parent strain and themutated viruses arising from the parent strain resultedin sequence differences of less than one percent(Chang et al., 2002; Rowland et al., 1999). This is incontrast to sequence differences of >15% commonlyseen in the field. On this basis of the experimentalevidence, diagnostic laboratories providing PRRSsequencing results consider sequence changes greaterthan 0.5 to 1.0 percent to be suggestive of aninterpretation that two viruses are not closely related(Christopher-Hennings et al., 2002). Althoughsequencing is the best tool to assess the relatedness ofstrains, more definitive statements generally cannotbe provided. That is, the origin, disease causingpotential, or biological properties of a field isolatecannot yet be predicted on the basis of genomicsequence information. Likewise, similarity of thegenomic sequence between field isolates andvaccines may not be an accurate predictor of efficacyand should not be used in selecting a vaccine.

References

AASP subcommittee on PRRS. 1996. Laboratory diagnosis of porcine reproductive and respiratory syndrome(PRRS) virus infection. Swine Health and Production 4(1):33-35.

Albina E, Leforban Y, Baron T, et al. 1992. An enzyme linked immunosorbent assay (ELISA) for the detection ofantibodies to the porcine reproductive and respiratory syndrome (PRRS) virus. Ann Rech Vet 23:167-176.

Albina E, Madec F, Cariolet R, et al. 1994. Immune response and persistence of the porcine reproductive andrespiratory syndrome virus in infected pigs and farm units. Vet Rec 134:567-573.

Allan GM, Ellis JA. 2000. Porcine circoviruses: a review. J Vet Diagn Invest 12:3-14.Andreyev V, Wesley R, Mengeling W, et al. 1997. Genetic variation and phylogenetic relationships of 22 porcine

reproductive and respiratory syndrome virus (PRRSV) field strains based on sequence analysis of open readingframe 5. Arch Virol 142:993-1001.

Baron T, Albina E, Leforban Y, et al. 1992. Report on the first outbreaks of the porcine reproductive andrespiratory syndrome (PRRS) in France: diagnosis and viral isolation. Ann Rech Vet 23:161-166.

Bautista EM, Goyal SM, Collins JE. 1993a. Serological survey for Lelystad and VR-2332 strains of porcinereproductive and respiratory syndrome (PRRS) virus in US swine herds. J Vet Diagn Invest 5:612-614.

Bautista EM, Goyal SM, Yoon IJ, et al. 1993b. Comparison of porcine alveolar macrophages and CL2621 for the

63

detection of porcine reproductive and respiratory syndrome (PRRS) virus and anti-PRRS antibody. J VetDiagn Invest 5:163-165.

Benfield D, Nelson E, Christopher-Hennings J. 1994a. Porcine reproductive and respiratory syndrome (PRRS)viral proteins and antigenic variation. Proceedings of the International Pig Veterinary Society Congress, p. 62.

Benfield DA, Nelson E, Collins JE, et al. 1992b. Characterization of swine infertility and respiratory syndrome(SIRS) virus (isolate ATCC VR-2332). J Vet Diagn Invest 4:127-133.

Benfield DA, Nelson EA, Christopher-Hennings J, et al. 1994b. Recent advances on the pathogenesis of PRRS.Proceedings of the Livestock Conservation Institute, p. 113-116.

Benson J, Yaeger M, Christopher-Hennings J, et al. 2002. A comparison of virus isolation, immunohistochemistry,fetal serology and reverse-transcription polymerase chain reaction assay for the identification of porcinereproductive and respiratory syndrome virus transplacental infection in the fetus. J Vet Diagn Invest 14:8-14.

Bierk M, Dee S, K Rossow et al. 2001. Diagnostic investigation of chronic porcine reproductive and respiratorysyndrome virus in a breeding herd of pigs. Vet Record 148:687-690.

Carlson DL, Fang Y, Nelson EA, Christopher-Hennings J. 2002. Discriminating between PRRSV isolates andvaccine with quantitative, real-time RT-PCR. Proceedings of the American Association of VeterinaryLaboratory Diagnosticians (in press).

Chang C-C, Yoon K-J, Zimmerman JJ, et al. 2002. Evolution of porcine reproductive and respiratory syndromevirus (PRRSV) during sequential passages in pigs. J Virol 76:4750-4763.

Chen Z, Plagemann PG. 1995. Detection of related positive-strand RNA virus genomes by reversetranscription/polymerase chain reaction using degenerate primers for common replicase sequences. Virus Res39:365-375.

Cho HJ, Deregt D, Joo HS. 1996. An ELISA for porcine reproductive and respiratory syndrome: production ofantigen of high quality. Can J Vet Res 60:89-93.

Christianson W and Joo HS. 1994. Porcine reproductive and respiratory syndrome: a review. Swine Health andProduction 2(2):10-28.

Christianson WT, Choi CS, Collins JE, et al. 1993. Pathogenesis of porcine reproductive and respiratory syndromevirus infection in mid-gestation sows and fetuses. Can J Vet Res 57:262-268.

Christianson WT, Collins JE, Benfield DA, et al. 1992. Experimental reproduction of swine infertility andrespiratory syndrome in pregnant sows. Am J Vet Res 53:485-488.

Christopher-Hennings J, Faaberg KS, Mengeling WL, et al. Porcine reproductive and respiratory syndrome (PRRS)diagnostics: Interpretation and limitations. J Swine Health Prod 10(5):213-218.

Christopher-Hennings J., Holler LD, Benfield DA, Nelson EA. 2001. Detection and duration of porcinereproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissuesfrom Yorkshire, Hampshire and Landrace boars. J Vet Diagn Invest 13:133-142.

Christopher-Hennings J, Nelson EA, Benfield DA. 1996. Detecting PRRSV in boar semen. Swine Health andProduction 4:37-39.

Christopher-Hennings J, Nelson EA, Hines RJ et al. 1995b. Persistence of procine reproductive and respiratorysyndrome virus in serum and semen of adult boars. J Vet Diagn Invest 7:456-464.

Christopher-Hennings J, Nelson EA, Nelson JK, et al. 1995a. Detection of porcine reproductive and respiratorysyndrome virus in boar semen by PCR. J Clin Microbiol 33:1730-1734.

Christopher-Hennings J., Nelson EA, Nelson JK, et al. 1998. Identification of porcine reproductive and respiratorysyndrome virus in semen and tissues from vasectomized and nonvasectomized boars. Vet Pathol 35:260-267.

Cook CA, Spatz SJ. 1998. Development of a commercial diagnostic assay for the detection of PRRSV in bodyfluids. Proceedings of the American Association of Swine Practitioners, pp. 267-274.

Dea S, Bilodeau R, Athanassious R, et al. 1992. Swine reproductive and respiratory syndrome in Quebec: isolationof an enveloped virus serologically-related to Lelystad virus. Can Vet J 33:801-808.

Dea S, Gagnon CA, Mardassi H, Milane G. 1996. Antigenic variability among North American and Europeanstrains of porcine reproductive and respiratory syndrome virus as defined by monoclonal antibodies to thematrix protein. J Clin Microbiol 34:1488-1493.

64

Dee SA, Bierk MD, Deen J, Molitor TW. 2001a. An evaluation of test and removal for the elimination of porcinereproductive and respiratory syndrome virus from 5 swine farms. Can J Vet Res 65:22-27.

Dee S, Torremorell M, Rossow K et al., 2001b. Identification of genetically diverse sequences (ORF 5) of porcinereproductive and respiratory syndrome virus in a swine herd. Can J Vet Res 65:254-260.

Dewey C, Charbonneau G, Carman S, et al. 2000. Lelystad-like strain of porcine reproductive and respiratorysyndrome virus (PRRSV) identified in Canadian swine. Can Vet J 41: 493-494.

Done SH, Paton DJ, White ME. 1996. Porcine reproductive and respiratory syndrome (PRRS): a review, withemphasis on pathological, virological and diagnostic aspects. Br Vet J 152:153-174.

Done SH, Paton DS, Edwards S, et al. 1992. Porcine reproductive and respiratory syndrome (“blue-eared” pigdisease). Pig Vet J 28:9-23.

Drew TW, Meulenberg JJM, Sands JJ, Paton DJ. 1995. Production, characterization and reactivity of monoclonalantibodies to porcine reproductive and respiratory syndrome virus. J Gen Virol 76:1361-1369.

Drew TW. 1995. Comparative serology of porcine reproductive and respiratory syndrome in eight Europeanlaboratories, using immunoperoxidase monolayer assay and enzyme-linked immunosorbent assay. Rev SciTech OIE 14:761-775.

Edwards S, Robertson IB, Wilesmith JW, et al. 1994. PRRS (“blue-eared pig disease”) in Great Britain. AmericanAssociation of Swine Practitioners Newsletter 4:32-36.

Egli C, Thür B, Liu L, et al. 2001. Quantitative TaqMan RT-PCR for the detection and differentiation of Europeanand North American strains of porcine reproductive and respiratory syndrome virus. J Virol Methods 98:63-75.

Ferrin N, Fang Y, Carroll J, et al. 2002. Validation of a blocking ELISA for antibodies against PRRSV.Proceedings of the International Pig Veterinary Society Congress, p. 365.

Frey ML, Eernisse KA, Landgraf JG, et al. 1992. Diagnostic testing for SIRS virus at the National VeterinaryServices Laboratories (NVSL). American Association of Swine Practitioners Newsletter 4:31.

Gilbert SA, Larochelle R, Magar R, et al. 1997. Typing of porcine reproductive and respiratory syndrome virusesby a multiplex PCR assay. J Clin Microbiol 35:264-267.

Goyal SM, Collins J. 1992. SIRS serology and virus isolation. Minnesota Board of Animal Health Newsletter37:2.

Goyal SM. 1993. Porcine reproductive and respiratory syndrome. J Vet Diagn Invest 5:656-664.Halbur P, Andrew J, Paul P, et al. 1995a. Strain variation of PRRS virus: field and research experiences.

Proceedings of the American Association of Swine Practitioners, pp. 200-204.Halbur PG, Andrews JJ, Huffman EL, et al. 1994. Development of a streptavidin-biotin immunoperoxidase

procedure for the detection of porcine reproductive and respiratory syndrome virus antigen in porcine lung. JVet Diagn Invest 6:254-257.

Halbur PG, Miller LD, Paul PS, et al. 1995b. Immunohistochemical identification of porcine reproductive andrespiratory syndrome virus (PRRSV) antigen in the heart and lymphoid systems of three-week-old colostrum-deprived pigs. Vet Pathol 32:200-204.

Halbur PG, Paul PS, Janke BH. 1993. Viral contributors to the porcine respiratory disease complex. Proceedingsof the American Association of Swine Practitioners, pp. 343-350.

Haynes JS, Halbur PG, Sirinarumitr T, et al. 1997. Temporal and morphologic characterization of the distributionof porcine reproductive and respiratory syndrome virus (PRRSV) by in situ hybridization in pigs infected withisolates of PRRSV that differ in virulence. Vet Pathol 34:39-43.

Henry SC. 1994. Clinical considerations in "acute" PRRS. Proceedings of the American Association of SwinePractitioners, pp. 231-235.

Houben S, Callebaut P, Pensaert MB. 1995a. Comparative study of a blocking enzyme-linked immunosorbentassay and the immunoperoxidase monolayer assay for the detection of antibodies to the porcine reproductiveand respiratory syndrome virus in pigs. J Virol Methods 51:125-128.

Houben S, van Reeth K, Pensaert MB. 1995b. Pattern of infection with the porcine reproductive and respiratorysyndrome virus on swine farms in Belgium. J Vet Med [B] 42:209-215.

65

Horter DC, Pogranichniy RM, Chang C-C, et al. 2002. Characterization of the carrier state in porcine reproductiveand respiratory syndrome virus infection. Vet Microbiol 86:213-228.

Joo HS. 1993. PRRS: Diagnosis. Proceedings of the Allen D. Leman Swine Conference, pp. 53-55.Kapur V, Elam MR, Pawlovich TM, Murtaugh MP. 1996. Genetic variation in porcine reproductive and respiratory

syndrome virus isolates in the Midwestern United States. J Gen Virol 77:1271-1276.Keay S, Moreau IA, Provis P. 2002. A retrospective study of PRRS diagnostic results from routine serologic

testing of seven PRRS naïve herds. Proceedings of the American Association of Swine Veterinarians, pp. 147-148.

Keffaber KK, Stevenson GW, Van Alstine WG, et al. 1992. SIRS virus infection in nursery/grower pigs.American Association of Swine Practitioners Newsletter 4:38-40.

Keffaber KK. 1989. Reproductive failure of unknown etiology. American Association of Swine PractitionersNewsletter 1:1-9.

Key K, Guenette DK, Yoon K-J, et al. 2002. Identification and differentiation of vaccine-like isolates of porcinereproductive and respiratory syndrome virus from field isolates using a heteroduplex mobility assay. J ClinMicrobiol (in review).

Key K, Haqshenas G, Guenette D, et al. 2001b. Genetic variation and phylogenetic analyses of the ORF 5 gene ofacute porcine reproductive and respiratory syndrome virus isolates. Vet Microbiol 83:249-263.

Kleiboeker SB, Lehman JR, Fangman TJ. 2002. Evaluation of RT-PCR on oropharyngeal scrapings and pairedserology for detection of PRRSV in sows. J Swine Health Prod (in press).

Larochelle R, Mardassi H, Dea S, Magar R. 1996. Detection of porcine reproductive and respiratory syndromevirus in cell cultures and formalin-fixed tissues by in situ hybridization using a digoxigenin-labeled probe. JVet Diagn Invest 8:3-10.

Legeay O, Bounaix S, Denis M, et al. 1997. Development of a RT-PCR test coupled with a microplate colorimetricassay for the detection of a swine Arterivirus (PRRSV) in boar semen. J Virol Methods 68:65-80.

Mardassi H, Wilson L, Mounir S, Dea S. 1994. Detection of porcine reproductive and respiratory syndrome virusand efficient differentiation between Canadian and European strains by reverse transcription and PCRamplification. J Clin Microbiol 32:2197-2203.

Mendez-Trigo A. 1993. PRRS serologic and virus isolation observations at the diagnostic center of Oxfordveterinary laboratories. Proceedings of the Livestock Conservation Institute, pp. 100-101.

Meng XJ. 2000. Heterogeneity of porcine reproductive and respiratory syndrome virus: implications for currentvaccine efficacy and future vaccine development. Vet Microbiol 74:309-329.

Mengeling WL, Lager KM, Vorwald AC. 1995. Diagnosis of porcine reproductive and respiratory syndrome. JVet Diagn Invest 7:3-16.

Mengeling WL, Lager KM, Vorwald AC. 1996a. Alveolar macrophages as a diagnostic sample for detectingnatural infection of pigs with porcine reproductive and respiratory syndrome virus. J Vet Diagn Invest 8:238-240.

Mengeling WL, Paul PS, Lager KM. 1993. Virus-induced maternal reproductive failure of swine. J Am Vet MedAssoc 203:1268-1272.

Mengeling WL, Vorwald AC, Lager KM, Brockmeier SL. 1996b. Diagnosis of porcine reproductive andrespiratory syndrome using infected alveolar macrophages collected from live pigs. Vet Microbiol 49:105-115.

Minehart MJ, Nelson EA, Harley DJ, et al. 1992. Comparison of virus neutralization (VN) and indirect fluorescentantibody (IFA) for the detection of antibodies to porcine reproductive and respiratory syndrome (PRRS) virus.Proceedings of the Conference for Research Workers in Animal Disease, p. 66.

Morrison RB, Collins JE, Harris L, et al. 1992. Serological evidence incriminating a recently isolated virus (ATCCVR-2332) as the cause of swine infertility and respiratory syndrome (SIRS). J Vet Diagn Invest 4:186-188.

Murtaugh M, Elam M, Kakach LT. 1995. Comparison of the structural protein coding sequences of the VR-2332and Lelystad virus strains of the PRRS virus. Arch Virol 140:1451-1460.

Nelson EA, Nelson JK, Couture LP, et al. 1997. A single amino acid substitution allows for the differentiation of

66

the PrimePac® PRRS vaccine from field isolates of PRRSV. Proceedings of the Conference for ResearchWorkers in Animal Disease, Abstr #202.

Nodelijk G, van Leengoed LAMG, Schoevers EJ, et al. 1997. Seroprevalence of porcine reproductive andrespiratory syndrome virus in Dutch weaning pigs. Vet Microbiol 56:21-32.

Nodelijk G, Wensvoort G, Kroese B, et al. 1996. Comparison of a commercial ELISA and an immunoperoxidasemonolayer assay to detect antibodies against porcine respiratory and reproductive syndrome virus. VetMicrobiol 49:285-295.

O’Connor M, Fallon M, O’Reilly PJ. 2002. Detection of antibody to porcine reproductive and respiratorysyndrome (PRRS) virus: reduction of cut-off value of an ELISA, with confirmation by immunoperoxidasemonolayers assay. Irish Vet J 55:73-75.

Ohlinger VF, Haas B, Sallmüller A, et al. 1992. In vivo and in vitro studies on the immunobiology of PRRS.American Association of Swine Practitioners Newsletter 4:24.

Oleksiewicz MB, Bøtner A, Madsen KG, et al. 1998. Sensitive detection and typing of porcine reproductive andrespiratory syndrome virus by RT-PCR amplification of whole viral genes. Vet Microbiol 64:7-22.

Park BK, Joo HS, Dee SA, Pijoan C. 1995. Evaluation of an indirect fluorescent IgM antibody test for the detectionof pigs with recent infection of porcine reproductive and respiratory syndrome virus. J Vet Diagn Invest7:544-546.

Paton DJ, Brown IH, Scott AC, et al. 1992a. Isolation of a Lelystad virus-like agent from British pigs and scanningelectron microscopy of infected macrophages. Vet Microbiol 33:195-201.

Paton DJ, Drew TW, Brown IH, et al. 1992b. Laboratory diagnosis of porcine reproductive and respiratorysyndrome. Pig Vet J 29:188-192.

Pirzadeh B, Gagnon C, Dea S. 1998. Genomic and antigenic variations of porcine reproductive and respiratorysyndrome virus major envelope GP5 glycoprotein. Can J Vet Res 62:170-177.

Rossow KD, Shivers JL, Yeske PE. 1999. Porcine reproductive and respiratory syndrome virus infection inneonatal pigs characterized by marked neurovirulence. Vet Record 144:444-448.

Rossow KD. 1998. Porcine reproductive and respiratory syndrome. Vet Pathol 35:1-20.Rossow KD, Bautista EM, Goyal SM, et al. 1994. Experimental porcine reproductive and respiratory syndrome

virus infection in one-, four-, and 10-week-old pigs. J Vet Diagn Invest 6:3-12.Rowland RRR, Benfield DA, Steffen M. 1996. PRRS virus replication in peripheral blood mononuclear cells

during persistent infection. Proceedings of the Conference for Research Workers in Animal Disease, Abstr #189.Rowland RR, Steffen M, Ackerman T, Benfield DA. 1999. The evolution of porcine reproductive and respiratory

syndrome virus: quasispecies and emergence of a virus subpopulation during infection of pigs with VR-2332.Virology 259:262-266.

Shin J, Torrison J, Choi CS, et al. 1997. Monitoring of porcine reproductive and respiratory syndrome virusinfection in boars. Vet Microbiol 55:337-346.

Spagnuolo-Weaver M, Walker IW, Campbell ST, et al. 2000. Rapid detection of porcine reproductive andrespiratory syndrome viral nucleic acid in blood using a fluorimeter based PCR method. Vet Microbiol 76:15-23.

Spagnuolo-Weaver M, Walker IW, McNeilly F, et al. 1998. The reverse transcription polymerase chain reactionfor the diagnosis of porcine reproductive and respiratory syndrome: Comparison with virus isolation andserology. Vet Microbiol 62:207-215.

Suarez P, Zardoya R, Prieto C, et al. 1994. Direct detection of the porcine reproductive and respiratory syndrome(PRRS) virus by reverse polymerase chain reaction (RT-PCR). Arch Virol 135:89-99.

Sur J-H, Cooper VL, Galeota JA, et al. 1996. In vivo detection of porcine reproductive and respiratory syndromevirus RNA by in situ hybridization at different times postinfection. J Clin Microbiol 34:2280-2286.

Swenson SL, Hill HT, Zimmerman J, et al. 1994. Excretion of porcine reproductive and respiratory syndrome virusafter experimentally induced infection in boars. J Am Vet Med Assoc 204:1943-1948.

Takikawa N, Kobayashi S, Ide S, et al. 1996. Detection of antibodies against porcine reproductive and respiratorysyndrome (PRRS) virus in swine sera by enzyme-linked immunosorbent assay. J Vet Med Sci 58:355-357.

67

Thacker BJ. 1992. Serological surveys in a herd before, during and after an outbreak of SIRS. AmericanAssociation of Swine Practitioners Newsletter 4:40.

Torremorell M, Polson D, Henry SC, Morrison RB. 2002. Specificity of the PRRSV IDEXX ELISA test innegative farms. Proceedings of the International Pig Veterinary Society Congress 1:209.

Umthun A, Mengeling W. 1999. Restriction fragment length polymorphism analysis of strains of porcinereproductive and respiratory syndrome virus by use of a nested-set reverse transcriptase-polymerase chainreaction. Am J Vet Res 60:802-806.

Van Alstine WG, Kanitz CL, Stevenson GW. 1993a. Time and temperature survivability of PRRS virus in serumand tissues. J Vet Diagn Invest 5:621-622.

Van Alstine WG, Stevenson GW, Kanitz CL. 1993b. Diagnosis of porcine reproductive and respiratory syndrome.Swine Health and Production 1(4):24-28.

Wensvoort G, de Kluyver EP, Luijtze EA, et al. 1992. Antigenic comparison of Lelystad virus and swine infertilityand respiratory syndrome (SIRS) virus. J Vet Diagn Invest 4:134-138.

Wensvoort G, Terpstra C, Pol JMA, et al. 1991. Mystery swine disease in the Netherlands: the isolation of Lelystadvirus. Vet Q 13:121-130.

Wesley RD, Mengeling WL, Lager KM, et al. 1998a. Differentiation of a porcine reproductive and respiratorysyndrome virus vaccine strain from North American field strains by restriction fragment length polymorphismanalysis of ORF 5. J Vet Diagn Invest 10:140-144.

Wills RW, Zimmerman JJ, Yoon K-J, et al. 1997a. Porcine reproductive and respiratory syndrome virus: apersistent infection. Vet Microbiol 55:231-240.

Yang L, Frey ML, Yoon K-J, et al. 2000. Categorization of North American porcine reproductive and respiratorysyndrome viruses: Epitopic profiles of the 15, 19, 25 and 45 kD proteins and susceptibility to neutralization.Arch Virol 145:1599-1619.

Yang L, Yoon K-J, Li Y, et al. 1999. Antigenic and genetic variations of the 15kD nucleocapsid protein of porcinereproductive and respiratory syndrome virus isolates. Arch Virol 144:525-546.

Yoon IJ, Joo HS, Christianson WT, et al. 1992a. An indirect fluorescent antibody test for the detection of antibodyto swine infertility and respiratory syndrome virus in swine sera. J Vet Diagn Invest 4:144-147.

Yoon IJ, Joo HS, Christianson WT, et al. 1992b. Isolation of a cytopathic virus from weak pigs on farms with ahistory of swine infertility and respiratory syndrome. J Vet Diagn Invest 4:139-143.

Yoon IJ, Joo HS, Christianson WT, et al. 1993. Persistent and contact infection in nursery pigs experimentallyinfected with porcine reproductive and respiratory syndrome (PRRS) virus. Swine Health and Production1(4):5-8.

Yoon K-J, Chang C-C, Zimmerman JJ, Harmon KM. 2001. Genetic and antigenic stability of PRRS virus inpersistently infected pigs: clinical and experimental prospectives. Adv Exp Med Biol 494:25-30.

Yoon K-J, Henry SC, Zimmerman JJ, Platt KB. 1996a. Isolation of porcine cytomegalovirus (PCMV) from a swineherd with PRRS. Vet Med 91:779-784.

Yoon K-J, Wu L-L, Zimmerman JJ, Platt KB. 1997. Field isolates of porcine reproductive and respiratorysyndrome virus (PRRSV) vary in their susceptibility to antibody dependent enhancement (ADE) of infection.Vet Microbiol 55:277-287.

Yoon K-J, Zimmerman JJ, McGinley MJ, et al. 1995a. Failure to consider the antigenic diversity of porcinereproductive and respiratory syndrome (PRRS) virus isolates may lead to misdiagnosis. J Vet Diagn Invest7:386-387.

Zeman D, Neiger R, Yaeger M, et al. 1993. Laboratory investigation of PRRS virus infection in three swine herds.J Vet Diagn Invest 5:522-528.

Zhou E, Zimmerman JJ, Zhou KX, et al. 2001. Development of a blocking ELISA for detection of swine antibodiesto PRRSV. Proceedings of the American Association of Veterinary Laboratory Diagnosticians, p. 60.