porcine reproductive and respiratory syndrome

4 th International Symposium on Emerging and Re-emerging Pig Diseases – Rome June 29 th – July 2 nd , 2003 

ADVANCES IN DIAGNOSTIC TECHNIQUES 

S. Belák, P. Thorén, M. Hakhverdyan 

The National Veterinary Institute, Research and Development Section, Department of Virology, Biomedical Centre, Uppsala, Sweden 

Abstract 

The application of genetic-based systems for the direct 

detection of viruses is discussed, with special regard to the 

different variants of the PCR. The group started the 

development of various PCR systems for routine diagnosis as 

early as in 1987-88. Subsequently, more than 60 PCR assays 

have been developed for routine diagnostic use. The first 

assays were classical PCR, nested-, or semi-nested PCR. 

Four commercial PCR kits were developed. Special 

instruments and laboratory practice have been introduced to 

avoid false positive results. False negatives are avoided by 

the use of internal controls of amplification (mimics). 

Recently, various methods of real-time PCR (TaqMan, 

Molecular Beacons, Primer-Probe Energy Transfer System) 

have been developed and are used in the routine diagnostic 

laboratory, i.e., for the detection and differentiation of swine 

vesicular virus and vesicular stomatitis virus. Multiplex PCR 

packages are under developed in the frame of a EC project, 

for the simultaneous detection of eight important viruses of 

swine: classical swine fever virus, African swine fever virus, 

porcine reproductive and respiratory syndrome virus, 

Aujeszky's disease virus, porcine parvovirus, swine vesicular 

disease virus, foot and mouth disease virus, and vesicular 

stomatitis virus. In order to further simplify and accelerate the 

work, nucleic acid extraction robot and pipetting robot are 

applied. Thus, PCR diagnostic procedures are rapid, robust 

and automated. By following the rules of the Office 

International des Epizooties (OIE), international consortia of 

laboratories are working on the five steps of validation and 

standardisation, including “ring tests”. 

Introduction 

The diagnosis of emerging and re-emerging viral diseases of 

animals and humans is rapidly improving today. Series of 

new techniques are developed both for the direct and for the 

indirect detection of viruses. Concerning direct detection, 

various molecular methods are introduced, like classical- 

PCR, real-time PCR, multiplex PCR, improved in situ 

hybridisation assays, in situ PCR. Further methods, like PCRrobotics, 

PCR for protein detection (DNA tags), novel nucleic 

acid hybridisation methods, improved sample enrichment, 

amplification without thermocycling, macro- and microarrays 

are under development as novel direct methods for today and 

for tomorrow. As far as indirect detection is concerned, also 

many approaches like recombinant proteins and new panels 

of monoclonal antibodies are used in ELISA systems, 

synthetic proteins, biosensors, bioluminometry, fluorescence 

polarisation, chemoluminescence are introduced. Both the 

direct and the indirect detection methods are subjects of 

simplification. Portable PCR machines, pen-side tests (like 

dip-sticks) are under development. The “in house” tests of the 

individual laboratories are replaced by standardised, 

validated assays. By following the rules of the Office 

International des Epizooties (OIE), international consortia of 

laboratories are working on the five steps of standardisation, 

including “ring tests”. The standardisation work is supported 

by various authorities, like OIE, European Commission (EC), 

the Food and Agricultural Organisation (FAO) and the 

International Atomic Energy Agency (IAEA). 

The international collaboration and standardisation of assays 

are highly required, if we consider that the globalised trade in 

live animals, animal products, bedding and feeds is leading to 

continuously increasing threat of infectious diseases world- 

Key words: SVDV, VSV, PCR, real-time PCR, multiplex PCR 

19 

wide. Ceasing of border-controls, like the internal borders of 

the European Union, and the increased traffic also 

contribute to an increased risk situation. Under such 

conditions the infectious agents may travel thousands of 

kilometres and suddenly appear in areas where they are 

unexpected and probably even unknown. The emerging 

agent may lead to improper or delayed diagnosis, which 

results in the uncontrolled spread of the infection to 

susceptible populations of animals in large unrestricted 

geographic areas. The recent outbreaks of foot-and-mouth 

disease (FMD) or classical swine fever (CSF) in several 

countries of Europe are examples, which have to be 

seriously regarded when the necessity of rapid and reliable 

diagnosis is discussed. 

In this article we summarise the developments and 

experiences of our group in the field of direct detection of 

viruses by using genetic-based systems, with special 

regards to the different variants of the PCR. General 

technical experiences will be discussed. Special attention 

will be paid to several important diseases of swine. 

PCR, semi-nested and nested PCR 

Our group, the Research and Development Section of the 

Department of Virology, National Veterinary Institute (SVA) 

was among the first ones to develop various PCR assays 

for the detection of viruses of animals and to introduce the 

methods in routine diagnosis. The work with diagnostic 

DNA methods started here in 1985 with the development of 

simple nucleic acid hybridisation assays, like the direct filter 

hybridisation assay (5, 6). The application of the PCR 

started in 1987-88, very soon after the description of the 

method (7). Since that time more than 60 new PCR assays 

have been developed for routine diagnostic use. The 

detailed description of the assays is given in articles, which 

are summarised in several reviews (8, 9, 10). 

Diagnostic PCR assays 

When developing these assays, the purpose was to obtain 

systems, which have a wide range and a high sensitivity of 

detection. In order to detect a wide range of viruses, the 

primers are selected from relatively conservative regions of 

the viral genome. First, “simple” PCR assays were 

developed, which were soon replaced by semi-nested or 

nested systems, providing higher specificity and sensitivity. 

Concerning swine viruses, the following nested PCR 

systems have been developed (the amplified region of the 

genome is shown in brackets), see also references Belák et 

al. (7); Vilcek et al. (32); Widén et al. (33); Stadejek et al. 

(25). 

DNA-viruses: Porcine parvovirus (VP2), Adenovirus 

(hexon), Pseudorabies (Aujeszky’s disease virus (gB, gE, 

gD), Porcine cytomegalovirus (DNA polymerase). 

RNA-viruses: Classical swine fever virus (NS3, E2), 

General pestivirus assay (5’NCR), Encephalomyocarditis 

virus (PP). 

The sensitivity of the nested PCR systems is very high, 1- 

10 genome copies of the target viruses are detected. 

Simultaneously, the nested PCR assays provide very high 

specificity. 

In general the systems amplify entirely the target virus(es). 

However, in certain cases the detection range is wide, 

according to the given diagnostic requirement. The widerange 

assays are practical to start screening of specimens


and the positive samples are further tested with more specific 

systems. Such wide range assays is the General pestivirus 

assays, which detects all three pestiviruses in swine, e.g., 

classical swine fever virus, bovine viral diarrhoea virus and 

border disease virus (ovine pestiviruses). The general assay 

is very useful to screen for the presence of pestiviruses and 

then three specific PCR assays determine the identity of the 

given pestivirus in the swine populations. The adenovirus 

(hexon gene) PCR is also general; it amplifies all mammalian 

adenoviruses, except for the members of the proposed 

Atadenovirus genus (Belák et al., in preparation). 

PCR assays for phylogeny 

In contrast to the diagnostic PCR assays, in this case the 

purpose is not a wide-range detection, but a high 

phylogenetic resolution. The sensitivity is necessary, but not 

as highly regarded as in the case of the diagnostic PCR 

assays. To provide high phylogenetic resolution, the variable 

genomic regions of the viral genomes are targeted. Such 

phylogeny PCR assays were developed for example to group 

pestiviruses (3’NCR, 5’NCR, E2, NS2, etc) (11, 12, 21, 22, 

23, 24, 28, 29, 30, 32). 

Molecular epizootiology 

By using the phylogeny PCR assays, one can not only group 

the viruses and determine the phylogenetic relations, but also 

rapidly identify the various variants of the virus. The genetic 

identification may be very exact and very rapid (within several 

days or hours) and this information can be crucial during 

various outbreaks. The spread of the various variants can be 

traced and the ways of spread may be cut rapidly, in order to 

prevent the distribution of the virus to large geographic areas 

and/or to large populations of susceptible animals. Thus, the 

rapid phylogenetic identification and tracing of the viruses is 

termed “molecular epizootiology”. Such studies were 

conducted, when genetic variant of classical swine fever virus 

(CSFV) were identified in several countries of Central Europe 

(24). Viral phylogeny and molecular epizootiology were also 

applied in a recent study when various variants of Porcine 

respiratory and reproductive syndrome virus (PRRSV) were 

partially sequenced and characterised. We determined ORF5 

sequences, representing pathogenic field strains from Poland 

and Lithuania, and currently available European-type live 

PRRSV vaccines. In addition, the complete ORF7 of 

Lithuanian and Polish strains was sequenced. The results 

showed that Polish, and in particular Lithuanian, PRRSV 

sequences were exceptionally different from the European 

prototype, the Lelystad virus, and in addition showed a very 

high national diversity. While all sequences were clearly of 

European type, inclusion of the new Lithuanian sequences in 

the genealogy resulted in a common ancestor for the 

European type virus significantly closer to the American-type 

PRRSV than previously seen. These observations provide 

support for the hypothesis that the EU and US genotypes of 

PRRSV evolved from a common ancestor (25). 

Molecular epizootiology provides a new way to trace the 

paths of the infectious agents and to combat the diseases. 

National and international tracing of virus variants helps to 

follow the spread of the infections and halts the spread to 

large geographic areas. Thus, the tools of molecular 

epizootiology are helping the veterinary authorities during the 

eradication programmes; with special regard to OIE List A 

diseases, like CSF and FMD. 

Real-time PCR assays 

Compared to “classical single or nested PCR”, the real-time 

PCR provides important advantages in the diagnostic 

laboratories (1, 2, 4, 19, 26, 27). Only one primer pair is used, 

but the system may still provide as high sensitivity level as 

the classical nested PCR. Simultaneously, the risk of 

20 

contamination is lower, since the system is closed and no 

sample-manipulation is needed (like PCR-product transfer 

in the nested PCR between amplification tubes, which is a 

severe risk of contamination). The contamination risk is 

further decreased by the fact that the fluorescence, 

indicating the results, is directly read through the unopened 

lid of the reaction vessel. Thus, there is no need to open the 

reaction vessels and there is no post-PCR manipulation to 

visualise the products. These procedures result in greatly 

reduced hands-on time, compared to previous PCR 

methods, where the products were run on agarose gels and 

the stained for visualisation. By eliminating the use of the 

potentially carcinogenic ethidium bromide stain, the health 

risk factors are also reduced. The single-run amplification 

the 96-well microtitre plate format allows automation of the 

PCR. The diagnostic work can further be automated by 

using robotics for nucleic acid extraction and pipetting. This 

is a crucial advantage of the real-time PCR assays, since 

automation will allow the similar easy use of PCR in 

diagnostic laboratories, like ELISA. Compared to previous 

amplification assays, the real-time PCR has a further 

advantage: it allows running quantitative PCR. Thus, the 

diagnostic answer is not only “yes” or “no”, but even the 

amount of the of the viral nucleic acids is determined, 

allowing calculations to estimate the viral load during 

infection (13). Such estimation opens new path not only for 

the diagnosis, but also for studying pathogenesis. The 

estimation of quantitative aspects is crucial when a virus 

commonly found in animals is possibly causing symptoms 

in relation to viral load, for example feline coronaviruses 

(15) or porcine circovirus 2 (PCV2; 17, 18). Comparative 

studies showed that because subclinical infections of pigs 

with PCV2 are common, the use of non-quantitative PCR as 

a diagnostic tool for PCV2-related diseases should be 

discouraged (20). The measurement of viral load is also 

important when estimating the effects of antiviral 

treatments, which is mainly exploited in the human 

medicine. 

Real-time PCR assays at our laboratory 

There is a range of real-time PCR chemistries to choose 

today, like TaqMan, molecular beacons (MB), scorpion 

primers, dual probe systems as utilized in the LighCycler® 

(Roche), dye-labelled oligonucleotide ligation (DOL), 

Primer-Probe Energy Transfer System (PriProET), etc. Our 

laboratory at NVI have developed diagnostic TaqMan, MB 

and PriProET systems and compared the applicability for 

the routine detection of viruses. These systems were first 

tested because a) the most reliable information was 

available for technical details; b) these systems seemed to 

be less sensitive for mutation rates of RNA viruses (an 

aspect important in diagnosis); c) the easy adaptability to 

our real-time PCR equipments, an ABI PRISM 7700 

Sequence Detector (Applied Biosystems) and Rotor-Gene 

2000 Real-Time Cycler (Corbett Research); d) relatively 

clear conditions of royalty and usage for diagnosis. 

TaqMan systems were developed at our laboratory or in 

collaboration with our partners for the detection of feline 

coronaviruses, parvoviruses, feline leukaemia virus, lactate 

dehydrogenase elevating virus, lymphocytic 

choriomeningitis virus, equine influenza, bovine 

coronavirus, bovine respiratory syncytial virus and equine 

rhinovirus. Furthermore, a “general” pestivirus TaqMan 

assay is also used (10). Molecular beacon (MB) assays 

were developed for swine vesicular disease virus and 

vesicular stomatitis virus, Indiana (Ind) and New-Jersey 

(NJ) serotypes (16). 

The detection level of these assays is around 10 genome 

copies, indicating very high analytical sensitivity. The 

assays were adapted for use in routine detection of viruses


and they allow diagnosis, within approximately four hours. 

The above listed real-time assays will be introduced in routine 

diagnosis as soon as the validation processes are finished 

(see below). 

The real-time PCR assays for detection of swine viruses are 

discussed in details below, considering emerging and reemerging 

diseases of swine as subjects of the present 

meeting. 

Real-time PCR assays for the detection of swine 

vesicular disease virus (SVDV) and vesicular stomatitis 

virus (VSV) using molecular beacons 

Due to the increasing incidence of viral vesicular diseases, 

the development of sensitive and type-specific systems is 

crucial to identify the viruses in the vesicular disease cluster 

of animals. MB probes have become powerful tools in 

diagnostic virology. MBs are single stranded oligonucleotide 

detector probes that form a stem-and-loop structure. MBs 

provide rapid and highly sensitive way of virus detection, 

allowing automated processes and reducing risk of 

contamination. Thus, our aim was the development of a rapid 

and sensitive detection method for SVDV and VSV of swine. 

A conservative region of SVDV 3D-gene and VSV L-gene has 

been chosen to design MB probes. Each MB was labelled 

with a differently coloured fluorophore: FAM for SVDV, JOE 

for VSV-NJ and ROX for VSV-Ind. DABCYL or Black Hole 

Quencher (Biosearch Technologies, Inc., USA) was used as 

a non-fluorescent quencher. Optimisation of the probes 

includes followings: a) Characterisation of the probe 

(calculation of the signal to background ratio and thermal 

denaturation profile); b) Sample dilution series; 3-5. Probe, 

primers and manganese titration series. An ABI PRISM 7700 

Sequence Detector was used for the experiments. 

At present SVDV MB is the most optimised among three 

designed probes. During the dilution series and generation of 

standard curve experiments (Fig. 1, 2, Table 1) the probe 

detected even 10 -6 diluted RNA templates. To develop an 

amplification control, cloning of SVDV PCR product is being 

carried out. SVDV MB probe specificity test shows successful 

amplification of all tested SVDV isolates, but not of human 

Coxsackie B5 virus (Fig. 3). VSV MBs need more 

optimisation experiments, especially, for VSV/NJ (16). 

Multiplex PCR 

The principle of the multiplex PCR is to use multiple primers to 

allow amplification of multiple templates within a single 

reaction. This is a very useful and practical idea for diagnostic 

purposes, providing the chance to detect more than one 

infectious agent in a single assay. The “classical” PCR 

techniques and the real-time PCR are equally suitable for 

designing broad-range, so called “multiplex” PCR assays. 

These assays have the capacity simultaneously detecting a 

panel of chosen microorganisms responsible from a given 

clinical case. For example, a single nasal specimen can be 

tested from an animal suffering from a respiratory disease, or 

a single rectal swab in the case of an enteritis/diarrhoea 

syndrome. The real-time PCR is more suitable for 

multiplexing, than the classical single or nested PCR, since 

here the individual probes for the component assays can be 

labelled with a number of different fluorophors, each of which 

functions as reporter dyes for one set of primers. Since the 

fluorescent probes emit different colour wavelengths, the realtime 

PCR is en excellent tool for the development of multiplex 

PCR assays. 

Multiplexing of the “classical nested” PCR techniques is rather 

complicated, considering the large number of oligonucleotides, 

which might interact with each other, as they are placed in the 

same reaction mix. In contrast, the concept of real-time PCR 

(using only single primer pairs) provides excellent possibilities 

for the construction of multiplex PCR assays with many 

21 

targets. 

EU project for the multiplex PCR detection of eight 

viruses of swine (QLK2-CT-2000-00486) 

In the frame of this ongoing project EU is supporting the 

development of multiplex PCR systems for the 

simultaneous detection of eight viruses of swine, including 

OIE List A viruses. The work is performed in collaboration of 

six European laboratories. 

The viruses to be studied are classical swine fever virus 

(CSFV), African swine fever virus (ASFV), porcine 

reproductive and respiratory syndrome virus (PRRSV), 

Aujeszky's disease virus (ADV), porcine parvovirus (PPV), 

swine vesicular disease virus (SVDV), foot and mouth 

disease virus (FMDV), and vesicular stomatitis virus (VSV). 

Multiplex PCR assays are under construction to detect 

clusters of viruses based on possible clinical presentation. 

The clusters will be: a) Respiratory (CSFV, ASFV, PRRSV, 

ADV); b) Reproductive (CSFV, ASFV, PRRSV, ADV, PPV); 

c) List A, haemorrhagic (CSFV, ASFV); d) List A, vesicular 

(SVDV, FMDV, VSV). 

The objectives are: 1. Development, standardisation and 

harmonisation of "conventional" gel-based PCR tests for 

detection of virus infections in swine. 2. Development, 

standardisation and harmonisation of fluorimeter-based 

real-time multiplex PCR tests for detection of virus 

infections in swine. 3. Development of multiplex nucleic acid 

enrichment procedures to increase sensitivity of multiplex 

PCR. 4. Development of methodology for multiplex 

detection of viral nucleic acid without thermocycling (i.e. 

Invader technique) using a DNA model. 5. Production and 

application of a library of internal controls for PCR 

technology to be applied to the above tests. These controls 

should allow EU wide standardisation and harmonisation of 

this technology. Further details see on: 

http://www.multiplex-eu.org/ 

At our laboratory several multiplex assays are under 

development. Respiratory viruses of horses (equine 

arteritvirus, equine herpesvirus 1 and 4, equine rhinovirus 2 

and equine influenza virus) and a general multiplex virus 

assay for detection of common viruses of cats (feline 

coronavirus, feline parvovirus and feline leukemia virus) are 

two examples. 

Robotics in nuclei acid extraction 

The speed and efficiency of the diagnosis is further 

increased by the use of a nucleic acid extraction robot 

(GenoVision, Norway, today Qiagen). This robot utilizes 

magnetic separation of the target molecules. We have 

compared the results of nucleic acid preparations of the 

robot with manual procedures and found the robot to be 

more efficient and precise (manuscript in preparation). In 

the robot, viral nucleic acids are purified simultaneously 

from 48 samples and the procedure is finished within 2.5 

hours. The products are clean enough to be amplified 

directly in the PCR. 

Automated diagnosis 

The simultaneous use of the robot and real time PCR 

detection provided an automated diagnostic procedure. 

With the introduction of a pipetting robot the automation is 

being now further completed. 

Precautions to avoid false positive results 

One risk factor in classical single or nested PCR is that false 

positive results, i.e., negative samples showing a positive 

reaction, may occur. This can be the reason of crosscontamination 

or product-carryover from positive samples. 

Such environmental contamination was a serious problem in 

the early days of PCR. Various methods and tools have


been tested in our laboratory to prevent false positive results. 

As a general practice today in PCR laboratories, samples and 

mixes are handled in laminar airflow hoods, which are 

regularly decontaminated using ultra-violet (UV-C) light and 

bleach, help to avoid false positives. We are running a 

recommended laboratory practice for the basic steps of nested 

PCR (mix and primer preparation, sample preparation, first 

and second PCR) in separate laboratory locations. In addition, 

special tube-holders and openers were constructed to 

minimise the false positive PCR results (7, 8, 10). 

Internal controls to avoid false negative results 

It is well documented today that inhibitory effects of 

ingredients, like heparin, semen components and other 

sample contaminants and/or pipetting errors might lead to 

false negative results of the PCR. In such cases the infected 

samples tested as negative. To avoid such misleading results, 

the use of internal controls (termed "mimics") is 

recommended. The mimics are safe indicators of amplification 

efficiency. A general rule for mimic construction is to produce 

nucleic acid molecules, which are different from the target viral 

nucleic acid, both in composition and in size, but having the 

same primer-binding sequences. Due to the same primerbinding 

nucleotide sequences, template and mimic are coamplified 

in the same tube without competition. The size 

differences between target and mimic provide an easy way to 

discriminate the true product from the mimic (3, 10). An 

alternative, particularly when working with RNA viruses, is to 

use a second primer set in the reaction, which is specific for 

the mRNA of a cellular “housekeeping” gene, which is 

constitutively expressed in all cells. 

Controls in real-time PCR 

When running real-time PCR assays, it is also important to 

incorporate internal controls. A practical approach is when a 

selected fragment of the host animal genome is co-amplified 

as an internal control. By including such an intrinsic control 

with its specific reporter fluorophore we obtain information on 

the sample quality and on pipetting errors. Simultaneously, 

the system shows the amplification of the target nucleotide 

sequences and provides safety for the diagnosis. 

Validation, standardisation 

The work of test validation and standardisation is extremely 

important today. Both national and international authorities 

require rigorous proof that the diagnostic assays are as 

reliable as possible. It is clear today that validation and 

standardisation are practical necessities as a “performance 

benchmark” if better new and more reliable diagnostic tests 

are to be developed and brought into everyday use. 

International agencies like the OIE, the Joint FAO/IAEA 

Division, national research institutions and commercial 

companies make great efforts to agree on international 

standardisation (34, 35). Considering these requirements, our 

laboratory (together with our partner institutions in Europe) 

has started the validation and standardisation of the routine 

diagnostic PCR assays. 

Diagnostic assay validation 

To make predictions about the performance of a diagnostic 

method, it is necessary to validate the assay in question. 

Validation is the evaluation of the method with the purpose to 

determine how fit the assay is for a particular field of use. 

General requirements for the competence of testing and 

calibration laboratories (EN ISO/IEC 17025:2000) 

This standard is from late 1999 and is the standard to be 

followed for accredited European laboratories performing 

routine diagnostic work and it substituted the standard EN 

45001:1989. Basically this standard gives the frame for the 

22 

work of an accredited laboratory and it specifies many 

important parameters in such an environment. Part of the 

standard is covering issues of validation. It is stated that the 

laboratory should validate: non-standardized methods, inhouse 

developed methods and standardized methods if 

they are used outside the original area of use. The 

validation process, routines and results should be 

documented and finally a statement by the laboratory 

discussing the suitability of the test can be made. The 

amount and quality of the work involved in such a validation 

process is largely determined by the needs of the 

customers. Examples of estimated parameters can be: LoD 

(Level of Detection), linearity of the method, reproducibility, 

repeatability, robustness or any other parameter interesting 

to the customer. This, rather general, standard has been 

further developed for the veterinary field by OIE. 

The OIE principles of validation 

OIE has published (2000; new version is due 2004) a 

standard for the validation of diagnostic assays in the 

veterinary field. Chapter I.3 in this standard (“Principles of 

validation of diagnostic assays for infectious disease”) 

describes in detail how to perform validation of the 

diagnostic assays in a standardised way. Since this chapter 

is of major importance, we provide here shortly the main 

points of assay validation according to OIE. 

Stage 1. Feasibility studies 

The first step in validating a new assay is to perform some 

kind of feasibility study. The aim is to determine whether or 

not a new assay is suitable to detect a range of virus 

concentrations without background activity. Several control 

samples (


assay. 

Precision and accuracy 

Repeatability and reproducibility are both important 

parameters of the assay precision. Repeatability is measured 

as both the amount of agreement between replicates within 

the same run or between replicates tested in different runs. 

Reproducibility is determined in several laboratories using the 

identical assay (protocol, reagents and controls). 

Accuracy is the amount of agreement between a test value 

and the expected value for a sample of known virus 

concentration. 

Stage 4. Monitoring validity of assay performance 

Estimation of the prevalence of a virus in the population is 

necessary for calculating the predictive value of positive 

(PV+) or negative (PV-) test results. 

D-SN and D-SP are rarely estimated in a proper way, which 

leads to a lack of good estimates of PV+ or PV-. Since this is 

extremely important information for judging the real 

performance of an assay when used in the field, it is 

advisable to change this shortcoming in the future. 

Stage 5. Maintenance and enhancement of validation criteria 

When the assay is accepted and used as a routine test it is 

important to maintain the quality control. Consistent 

monitoring for repeatability and accuracy is necessary. 

Reproducibility between laboratories (ring tests) is 

recommended by OIE to be estimated at least twice a year. 

With our partner laboratories we have performed series of 

ring test to validate the PCR assays for the detection of 

classical swine fever virus (21, 23). 

If the assay is to be applied in another geographic region, it 

might be necessary to revalidate it under the new conditions. 

Proper assay validation is time consuming and expensive. It 

is difficult to obtain suitable standard samples and a huge 

amount of samples are needed. It is not surprising that 

validation has been neglected or at least considered less 

important in the past. We can now see a clear trend that the 

same quality demands that has been used in human 

applications can now be found also in veterinary diagnostics. 

Validation is becoming one of the most important 

improvements of PCR, and other, diagnostics today. All 

laboratories which are seriously performing these services 

can no longer avoid it. This will for sure lead to safer and 

better assays in the future. 

Acknowledgements 

These studies have been carried out with financial support 

from the European Commission (QLK2-CT-2000-00486), The 

Swedish Farmer’s Foundation for Agricultural Research (SLF), 

The Swedish Council for Forestry and Agricultural Research 

(FORMAS) and by internal grants of the National Veterinary 

Institute. 

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Clin. Microbiol. 38, 3474-3477. 

19. McGoldrick, A., Lowings, P., Ibata, G., Sands, J., Belák, S., 

Paton, D. (1998). A novel approach to the detection of classical 

swine fever virus by RT-PCR with flurogenic probes (TaqMan). 

J. Virol. Methods 72, 125-135. 

20. McNeilly F, McNair I, O'Connor M, Brockbank S, Gilpin D, 

Lasagna C, Boriosi G, Meehan B, Ellis J, Krakowka S, Allan 

GM. (2002). Evaluation of a porcine circovirus type 2-specific 

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diagnosis of postweaning multisystemic wasting syndrome in 

pigs: comparison with virus isolation, immunohistochemistry, 

and the polymerase chain reaction. J. Vet. Diagn Invest, Mar. 

14(2), 106-112. 

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Classical swine fever virus: the second ring test to evaluate RT- 

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swine fever virus in Europe. Virus Res. 52, 195-204. 

25. Stadejek, T., Stankevicius, A., Storgaard, T., Oleksiewicz, M. 

B., Belák, S., Drew, T. W., Pejsak, Z. (2002). Identification of


radically different variants of porcine reproductive and respiratory 

syndrome virus (PRRSV) in Eastern Europe: Towards a common 

ancestor for European and American viruses. J. Gen. Virol. 83, 

1861-1873. 

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24 

31. Vilcek, S., Belák, S. (1998). Classical swine fever virus: 

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of the genome. Virus Genes 18, 107-114. 

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detection and determination of the putative partial sequence of 

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8.


PRRS 

Lectures 

25


26


MOLECULAR EPIDEMIOLOGY OF PRRSV 

T. Storgaard 1 , M. B. Oleksiewicz 1 , T. Stadejek 2 , R. Forsberg 3 , H. S. Nielsen 1 , and A. Bøtner 4 . 

1 Applied Trinomics, Preclinical Development, Novo Nordisk A/S, Måløv, Denmark; 2 National Veterinary Institute, Pulawy, Poland; 3 Department 

of Ecology and Genetics, University of Aarhus, Aarhus, Denmark; 4 Danish Veterinary Institute, Copenhagen, Denmark. 

Keywords: PRRS, epidemiology, emergence, genetic diversity, molecular clock 

The molecular epidemiology of porcine reproductive and respiratory 

syndrome virus is of major interest, both from a scientific 

and practical point of view. The almost simultaneous 

emergence in North America and Europe of genetically very 

distinct types of PRRSV is still a major scientific puzzle. We 

do not know from which reservoir(s) PRRSV emerged, nor do 

we know what triggered the emergence of clinical disease in 

pigs in the late 1980'ies. As such, we have no way of evaluating 

the risk of future emergence of new variants of PRRSV, 

and likewise, no chance of proactive development of diagnostic 

tests and vaccines. Maybe of more immediate practical 

relevance, and concern, to the swine industry is the observation 

that the genetic diversity of both currently known PRRSV 

genotypes is rapidly increasing, and has done so ever since 

the first complete-genome sequence of PRRSV was determined 

almost a decade ago. This rapid growth in the genetic 

diversity of the viral envelope glycoproteins is likely to be associated 

with reduced immunological cross protection. Examples 

of this may already exist, with reports that the existing 

vaccines appear to have poor effect against the new "acute" 

American-genotype PRRSV isolates. Also, with radical genetic 

changes being reported even in the PRRSV ORF7 (nucleocapsid) 

protein, which was hitherto though to be well 

conserved, and hence is widely used for diagnostic purposes, 

diagnostic laboratories may in the near future have to assure 

that their methods have the robustness required to handle the 

genetic diversity. Of even greater concern is that the ongoing 

genetic changes may result in PRRSV variants with fundamentally 

different biological properties. At the 3 rd symposium 

in Ploufragan, the state of the art was that high degree of 

genetic diversity existed for the North American-type of 

PRRSV, while in contrast, all European-type isolates were 

remarkably similar to the first European Lelystad–isolate. 

Several hypotheses could have explained such a scenario. 

For example, PRRSV could have acquired domestic pigs as a 

host in North America much earlier than was the case in 

Europe. That would have fitted with the observation that 

PRRSV antibodies had been detected in Canadian serum 

from 1979, and the fact that clinical disease was described 

several years earlier in North America than in Europe. However, 

at the symposium in Ploufragan, the first glimpse also 

appeared that the situation might potentially be just the opposite. 

An abstract described that very different European-type 

PRRSV isolates were found in Russia, and that these isolates 

apparently became gradually more different to the prototypic 

Lelystad isolate the further east they were obtained in Russia 

(1). Unfortunately, these data were never presented at the 

meeting in Ploufragan, nor have they to our knowledge been 

published or submitted to public sequence databases since 

then. The data were nevertheless very intriguing, and fitted 

nicely with the oral presentation given by Dr. Ohlinger in 

Ploufragan, who had found antibodies against PRRSV in East 

German pig sera originating from 1988. Taken together, 

these observations indicated the possibility of a scenario 

where PRRSV had gradually spread from Eastern to Western 

Russia over many years, then into Eastern Europe, from 

where it was finally transferred to Western Europe concurrent 

with the events surrounding the reunification of Germany. 

Since the Symposium in Plufragan, several studies have 

been published, which support a scenario like the above. It 

has been shown that the genetic diversity of Europeangenotype 

PRRSV in some European countries, for example 

27 

Denmark, Poland, Spain and Italy, is just as high as what is 

reported for American-genotype PRRSV from North America 

(2, 3, 4, 5). Even more intriguing, ORF5 and ORF7 sequences 

of three European-genotype PRRSVs from Lithuania 

have been published, that were dramatically different from all 

previously published European-genotype PRRSV ORF5 and 

7 sequences (6). So, with our current knowledge, it seems 

that PRRSV either acquired domestic pigs as hosts much 

earlier in Europe (or potentially eastern part of Russia) than 

was the case in North America, or alternatively, that several 

independent introductions of PRRSV from an unknown reservoir 

species into domestic pigs has taken place in Europe. 

Since Plufragan, we serendipitously discovered that the vast 

majority of the mutations identified in the ORF3 of Europeantype 

PRRSV appear to be neutral, and by that, accumulated 

by chance alone. Thus, ORF3 of European-type PRRSV can 

be used as a very precise molecular clock, with potential use 

for molecular epidemiology in the field (2, 5). For example, for 

European-type PRRSV transmitted from farm A to farm B, it is 

possible to estimate when transmission has occurred with a 

precision of 1-2 months, based solely on the ORF3 sequences 

obtained from the two farms. Likewise, the European-type 

ORF3 molecular clock can be used to date the 

introduction of European-type PRRSV into a given area or 

country. By phylogenetic analysis of ORF3 sequences from 

Danish, British, Dutch and Italian European-type PRRSV 

isolates, we were able to show that the most recent common 

ancestor for these isolates existed in 1979, more than ten 

years before the emergence of clinical disease in Europe. 

Furthermore, this analysis allowed us to estimate the rate of 

nucleotide substitutions to 6x10 -3 substitutions per site per 

year (2). This corresponds to the fastest rate of nucleotide 

substitutions reported for any glycoprotein of any RNA virus. 

It will of course be of utmost interest to obtain ORF3 sequences 

of the recently reported highly divergent Lithuanian 

isolates, to test if they also follow a molecular clock, and if so, 

to determine how long back in time they will push the age of 

the most recent common ancestor for European-type 

PRRSV. Unfortunately, we have not been able to detect a 

molecular clock in the ORF3 of North American-genotype 

PRRSV. Although still controversial, the literature suggests 

differences in the degree of virion-association between 

American-type and European-type ORF3 protein, and it may 

thus be that the selective pressures on ORF3 differ between 

North American-genotype and European-genotype PRRSV. 

Alternatively, genetic recombination could have confounded 

molecular clock analysis of American-genotype ORF3. We 

are in the process of collecting American-genotype vaccine 

isolates, and sequencing ORF3 from these, in order to test for 

a molecular clock. This work on PRRSV molecular clocks is 

driven by what we consider the current "holy grail" of PRRSV 

molecular epidemiology: dating the split between the North 

American and European genotypes, which should – for the 

first time – allow us to build a scientifically based hypothesis 

for the origin of PRRSV. Knowing the history and mechanisms 

of PRRSV evolution might not necessarily tell us the 

future of this virus, but it may contribute towards proactive 

surveillance, serodiagnostic and vaccine development 

schemes, akin to what is currently practiced for influenza virus.


References 

1. Andreyev, V.G., Scherbakov, A.G., Pylnov, V.A., and 

Gusev, A.A.: Genetic heterogeneity of PRRSV in Rusia. 

In Procedings of the Third International Symposium on 

Porcine Reproductive and Respiratory Syndrome (PRRS). 

pp 211-212. 

2. Forsberg, R., Oleksiewicz, M.B., Petersen, A.M.K., Hein, 

J., Bøtner, A., and Storgaard, T.: A molecular clock dates 

the common ancestor of European-type porcine reproductive 

and respiratory syndrome virus at more than 10 

years before the onset of the current epidemic. Virology, 

289: 174-179, 2001. 

3. Forsberg, R. Storgaard, T., Nielsen, H. N., Oleksiewicz, 

M. B., Cordioli, P., Sala, G., Hein, J., and Bøtner, A.: The 

genetic diversity of European type PRRSV is similar to 

that of the North American type but is geographically 

skewed within Europe. Virology, 299: 38-47, 2002. 

28 

4. Indik, S., Valicek, L., Klein, D., and Klanova, J.: Variations 

in the major envelope glycoprotein GP5 of Czech 

strains of porcine reproductive and respiratory syndrome 

virus. Journal of General Virology 81, 2497-2502, 2000. 

5. Oleksiewicz, M.B., Bøtner, A., Toft, P., Grubbe, T., Nielsen, 

J., Kamstrup, S., and Storgaard, T.: Emergence of 

porcine reproductive and respiratory syndrome virus 

(PRRSV) deletion mutants: correlation with the porcine 

antibody response to an antigenic site in the ORF3 structural 

glycoprotein. Virology 267:135-140, 2000. 

6. Stadejek, T., Stankevicius, A., Storgaard, T., Oleksiewicz, 

M. B., Belák, S., Drew, T., and Pejsak, Z.: Identification of 

radically different variants of porcine reproductive and 

respiratory syndrome virus (PRRSV) in Eastern Europe: 

Towards a common ancestor for European and American 

viruses. Journal of General Virology, 83: 1861-1873, 

2002.


PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME: CONTROL AND VACCINOLOGY 

Kelly M. Lager 

Virus and Prion Diseases of Livestock Research Unit, 

National Animal Disease Center, USDA, Agricultural Research Service, Ames, Iowa 50010, USA 

Porcine Reproductive and Respiratory Syndrome (PRRS) 

was first recognized about 16 years ago in the eastern United 

States as epidemics of maternal reproductive failure and 

severe respiratory disease in young pigs. At that time PRRS 

was known as Mystery Swine Disease (MSD) since its cause 

was unknown. Following the initial reports, MSD epidemics 

were quickly recognized throughout swine dense regions 

across the United States and Canada sparking some debate 

about whether the disease had spread rapidly from the 

original cases, or were veterinarians just becoming more 

aware of the clinical signs associated with MSD. This debate 

broadened with the first reports of MSD in Western Europe 

during the fall and winter of 1990. During this time there was 

much speculation about the cause of MSD although it was 

generally accepted that the etiology was infectious and the 

etiologic agent was most probably a virus. This assumption 

was confirmed in spring of 1991 when Dutch scientists 

discovered the virus that caused MSD or PRRS (64). This 

virus is now known as the PRRS virus (PRRSV) and it has 

been classified as a member of the Arteriviridae virus family 

for which equine arteritis virus is the prototype (40). By the 

early 1990s PRRS had become a pandemic, and to this day it 

is still causing significant losses for the swine industry. 

At the beginning of the PRRS story veterinarians and pork 

producers tried to fight this new disease with the tools that 

were available, common sense. Field observations 

suggested PRRS was an infectious disease and the 

assumption was the infectious agent causing PRRS would 

behave like any theoretical infectious agent; i.e., it would 

replicate in swine, it would be shed from infected swine for 

some period of time, it would spread among swine, and it 

would induce an immune response in swine that would 

protect them from clinical disease upon re-exposure. Using 

this logic, veterinarians implemented control plans in herds 

undergoing epidemics and prevention plans in herds deemed 

at risk of becoming affected by the disease. Basically, these 

control and prevention programs centered around the 

concept of exposing or immunizing naïve animals to the 

infectious agent through the use of cull animals (usually sows 

that had aborted) and the feedback of substances assumed 

to contain the infectious agent, e.g., feces, stillborn pigs, 

aborted fetuses, and placenta. In addition to these strategies, 

the practice of replacement-animal quarantine was 

encouraged. 

From the early days of MSD or PRRS it became apparent 

that the epidemiology of this disease was not simple. As the 

data from field studies began to accumulate, it appeared the 

MSD agent (PRRSV) could be transmitted through the use of 

contaminated semen collected from boar studs even though 

the boar studs in question may have been clinically normal 

(51). At times, direct spread of PRRSV indicated some sort 

of a chronic carrier state must exist for the virus. This 

hypothesis was based on the observation that swine could be 

moved from one herd that was clinically normal, although it 

had experienced a PRRS epidemic in the past, to another 

herd and the recipient herd would then develop PRRS. 

Indirect spread of PRRSV, i.e., spread of the virus not 

involving the direct contact of swine or the use of 

contaminated semen, was believed to occur. Another part of 

the epidemiology puzzle was an apparent cyclical form of the 

Key words: PRRSV, Control, Vaccinology 

29 

disease where herds underwent epidemics in a periodic 

pattern of every 9 to 18 months or so. All of these 

observations indicated the etiology of PRRS was complex 

and with the discovery of PRRSV, and the subsequent 

avalanche of PRRSV-related research, it became apparent 

that this virus was not a typical, theoretical infectious agent. 

In fact, this virus and the virus/host relationship have unique 

qualities that produce novel problems for the control and 

prevention of PRRS. 

The purpose of this paper about PRRS control and 

vaccinology is to review the past, comment on the present, 

and speculate about the future. Of course this paper will 

have a bias that is based on the experience of its author, a 

veterinary virologist working in a government laboratory 

located in the central United States. Before beginning a 

PRRS commentary, it is important to state a few general 

comments. First, despite all of the currently available 

technology and all that is known about PRRS, this disease 

still has plenty of mystery. Second, PRRS is a constant 

challenge for everyone in the swine industry trying to control, 

prevent, and eliminate this disease. Lastly, it is quite 

apparent that there is no simple answer to the PRRS problem 

and there seems to be few complex answers that work 

consistently in a herd over time. I will begin this paper with 

comments on epidemiology, immunology, and vaccines, 

followed by how this knowledge might be applied to control 

and prevent PRRS, and then conclude with speculation about 

the future. 

EPIDEMIOLOGY 

Etiologic agent - PRRSV is classified as a member of the 

Arteriviridae virus family in the order Nidovirales (13). Since a 

discussion of PRRSV properties is beyond the scope of this 

paper, only relevant information about the virus and how it 

relates to the control and prevention of PRRS will be 

discussed. 

Routes or Modes of Transmission 

Direct Transmission - Direct transmission defined as PRRSV 

transmitted between swine due to close contact or through 

the use of infected semen. 

When thinking about direct PRRSV transmission it is 

important to think of animals as young and old since the 

likelihood of virus transmission seems to be dependent on the 

age of the animal at the time of infection. For the sake of this 

paper, old swine will be defined as 6 to 7 months of age or 

greater. Young swine will be defined as about 4 weeks of 

age since most research involving pigs utilizes 3- to 5-weekold 

pigs. Keep in mind, my definitions are arbitrary and 

reflect research that has been published and my 

understanding of that research. Of course others may have a 

different opinion and one must always remember that when 

dealing with PRRS there are very few black and white issues. 

I believe the response of old swine to a PRRSV infection is 

the easiest to understand and the best place to start the 

discussion. 

Old Swine 

Susceptibility - Swine can be infected by oronasal, 

intravenous, intramuscular, intravaginal, and intratracheal


routes. They can be susceptible to as few as 100 CCID50 of 

wild-type PRRSV given intramuscularly (7). 

Viremia - Wild-type PRRSV infection will usually produce a 

viremia (detected by virus isolation) for a duration of less than 

3 weeks with a majority of old swine having a viremia lasting 

less than 2 weeks. 

Transmission - Horizontal PRRSV transmission from old 

swine to age-matched animals after 6 weeks of infection is 

unlikely under experimental conditions (5,33,34). However, 

there is one experimental report of PRRSV being transmitted 

99 days-post-infection from sows to finishing age swine (67). 

Interestingly, under field conditions PRRSV may or may not 

easily transmit among sows within a naïve herd (even when 

they share a common water trough). Why there are these 

apparent differences in transmission rates between naïve 

herds is unknown. Field evidence suggests chronic PRRSV 

shedding occurs at a low frequency in old swine. This 

statement is based on collective field evidence where a sow 

herd regains normal production following a PRRS epidemic 

and eventually many of the sows become seronegative and 

sentinels introduced into the sow herd do not become 

infected. The occurrence of PRRS-positive farms becoming 

PRRS-negative (22) suggests there are few sows in the herd 

that are chronically shedding PRRSV. These PRRS-positive 

herds that are clinically normal and eventually produce 

PRRS-negative pigs are referred to as PRRS-stable farms. 

As is the case all too frequently with PRRS, there are few, if 

any, absolute PRRS rules. Surely, there are times when 

sows do become infected with PRRSV and shed virus for 

extended periods of time. Perhaps these chronic shedders 

are what help keep some herds PRRS-unstable. To reinforce 

the comment "there are few, if any, absolute PRRS rules, one 

should keep in mind that subclinical PRRSV infections can 

occur in a herd. 

Vertical transmission is more likely to occur later in gestation 

than earlier and transplacental infection can occur within a 

week post-infection of the sow (14,29,30,35,36). PRRSV 

infection of the dam around the time of conception may have 

little direct impact on embryos, however, once they begin 

implantation, they may be more susceptible to PRRSV 

infection (49,50). 

Boars - Boars can shed PRRSV in semen for many weeks 

post-infection and in one case out to 92 days post-infection 

(15). Virus can be shed in semen intermittently, making 

negative semen tests from PRRSV-seropositive boars difficult 

to interpret. The quantity of virus shed in semen is not clear. 

This is an important issue if the amount of virus shed in 

semen is below the sensitivity of the test used to detect 

PRRSV in semen, yet this amount of virus is still infectious. 

The minimal infectious dose of PRRSV in semen for a sow is 

not known. 

Young Swine 

Susceptibility - Young swine also can be infected by the 

routes described above and pigs can be susceptible to as few 

as 20 CCID50 PRRSV given oronasally (66). 

Viremia - Wild-type PRRSV infection will usually produce a 

viremia (based on virus isolation) for a duration less than 7 

weeks, a majority will have a viremia lasting less than 5 

weeks. 

Transmission - Pigs infected with PRRSV have consistently 

shed virus up to 6 to 8 weeks-post-infection to age-matched 

pigs (57,62,65). One study reported virus transmission at 

about 22 weeks-post-infection to age-matched controls (1). 

30 

Congenitally infected pigs have shed virus to age-matched 

controls up to about 15 weeks-post-parturition (8). 

Persistence - In experimentally-infected pigs, PRRSV could 

be detected by virus isolation for 105 (27) and 150 days (2) 

post-infection and by PCR for up to 251 days post infection 

(63). Under field conditions it is assumed that PRRSV could 

persist in some pigs for even longer periods of time and these 

pigs could be a transmission risk. However, the significance 

of these animals as a transmission risk is unknown. In 

congenitally-infected pigs PRRSV nucleic acid was detected 

in the buffy coat for up to 230 days after parturition (8). In all 

of these studies that have detected virus or viral nucleic acid 

for extended periods of time post infection, the animals were 

always reported to be seropositive based on the methodology 

used in the respective laboratories. 

Immunotolerant - A PRRSV immunotolerant state may not 

exist in swine. Immunotolerance can be defined as a fetus 

that becomes infected with a pathogen early in gestation 

before the fetal immune system develops. The infection does 

not kill the fetus and as the fetal immune system develops the 

pathogen is recognized as normal fetal tissue. The fetus is 

born alive, is replicating the pathogen, sheds the pathogen for 

life, and has no detectable humoral immune response against 

the pathogen. Perhaps the best example of an 

immunotolerant state is the infection of the bovine fetus with 

bovine virus diarrhea virus (BVDV). The BVDV 

immunotolerant calf is a critical factor in the epidemiology of 

the disease since it is difficult to detect them and they shed 

virus to their penmates. In regards to PRRSV, a hypothetical 

immunotolerant pig could have tremendous impact in today's 

production systems where one pig could come into contact 

with thousands of pigs. 

I have only tested fetuses as young as about 40 days of age 

with wild-type or attenuated PRRSV infections and have not 

been able to prove that PRRSV can induce an 

immunotolerant state in pigs (32). Wild-type PRRSV 

eventually kills the fetuses, so little or no chance for the 

development of an immunotolerant state. Fetuses can be 

infected with attenuated PRRSV and they appear normal; 

however, they do develop a detectable immune response and 

the PRRSV-infected fetuses therefore are not 

immunotolerant. I think it would be a remote possibility that 

pigs could develop an immunotolerant state, i.e., a 

congenitally PRRSV-infected pig that does not develop a 

detectable humoral response to PRRSV and readily sheds 

PRRSV. Keep in mind that fetuses infected late in gestation 

can be born alive and may not have seroconverted by the 

time of parturition. However, if the pigs survive long enough, 

they do seroconvert. 

Indirect Transmission - Indirect transmission defined as 

PRRSV transmitted between swine that does not involve 

direct transmission. 

Possible modes of indirect transmission include aerosol, 

fomites, and vectors. Aerosol transmission is defined as the 

transfer of virus from one pig to another via movement of the 

virus in air. Field observations support this concept, perhaps 

over a considerable distance (28). Experimental 

observations support this concept over a distance of about 1 

meter (11,58). Fomites are objects that can be contaminated 

with a pathogen at one place and when moved to another site 

the pathogen is transferred, e.g., PRRSV-contaminated 

boots, gloves, needles, and semen-storage coolers 

(18,19,44). A vector would be a carrier of a pathogen from 

one pig or farm to another pig or farm. Mechanical vectors 

could be a PRRSV-contaminated creature, e.g., birds 

(carrying contaminated manure or feed on feet), blood-


sucking insects (transfer of virus contaminated blood) (45,46), 

animal (transfer of contaminated material on feet or fur) or 

people with dirty hands (44). Biological vectors are a 

creature that can actually replicate the pathogen and amplify 

the quantity of pathogen; in the case of PRRSV some 

waterfowl may be a biological vector (63). Current studies at 

the University of Minnesota are evaluating if mosquitoes can 

be a biologic vector for PRRSV. Mice and rats have not been 

susceptible to a PRRSV infection (26). 

Based on field observations and experimental studies, it 

seems that all of the previously described routes of indirect 

transmission are possible. The debate is about the 

probability of indirect PRRSV transmission by one of these 

routes and how to prevent that route of virus transmission. 

IMMUNOLOGY 

Although there is a growing body of research about PRRSV 

immunology, this area is still full of unknowns. At the pig level 

there are some consistent facts about the PRRSV-specific 

immune response that can be experimentally reproduced. 

However, these immune responses are not yet fully 

understood at the cellular and molecular levels. Of course, 

there are some presumed PRRSV-specific immune 

responses that are not understood completely at any level, 

specifically the debate over PRRSV-induced 

immunoregulation. Depending on methodology and the 

parameters tested, PRRSV may have inhibitory and/or 

stimulatory effects on the immune system that may or may 

not contribute to a synergism between PRRSV and other 

pathogens. Again, this subject is beyond the scope of this 

paper and for the sake of brevity, the PRRSV immune 

response will be thought of in a simplistic fashion. The focus 

of this section is on how the immune response can relate to 

control and prevention of PRRS. 

It is clear that swine experimentally infected with wild-type 

PRRSV develop a detectable humoral immune response 

within 7 to 10 days of infection (2,27,30,35,63). Peak 

antibody titers may occur by 5 to 6 weeks post infection and 

then the titer may slowly decline over the course of months to 

where the antibody levels are at the threshold of detection. A 

virus neutralizing antibody response develops about 4 weeks 

post infection and may be detectable for months. An 

interesting field observation that has been repeated in animal 

studies is the apparent lack of a classical anamnestic 

humoral immune response to a homologous PRRSV 

challenge, i.e., when the pig is exposed twice to the same 

virus there is no detectable rise in antibody titer after the 

second exposure (29-31). In addition, there is no clinical 

disease associated with the homologous challenge and no 

detectable replication of the homologous challenge virus 

except for one report where virus was only detected 3 days 

post challenge (53). 

When it comes to heterologous virus exposure, i.e., the 

second PRRSV exposure (challenge virus) is a different 

strain than the first PRRSV exposure (immunizing virus), a 

rise in antibody titer following challenge is usually detected 

(10,31,33,37-39,59). It seems reasonable that the postchallenge 

rise in antibody titer is in response to a set of 

different PRRSV antigens and this response is not a classical 

anamnestic immune response. Furthermore, it seems 

probable that the more antigenically related the PRRSV 

strains, the less likelihood that a heterologous challenge 

would induce a detectable humoral response. A lack of 

cross-protection following heterologous challenge can be 

characterized by an onset of clinical disease and/or the 

detection of challenge virus replicating in immunized swine. 

Cross-protection can be defined as no clinical disease, a 

31 

failure to detect replicating challenge virus, and no detectable 

rise in antibody titer. In regards to PRRSV, there is great 

genetic diversity, and presumably, antigenic diversity among 

PRRSV isolates. When it comes to control and prevention 

strategies, this diversity leads to many cross-protection 

questions that need to be answered. 

Serologic tests are a very important tool for use in control and 

prevention programs. In the United States a commercially 

available ELISA has performed well (PRRS HerdCheck, 

IDEXX Laboratories, Westbrook, Maine, USA) (52). 

According to the manufacturer, an ELISA S/P value equal to 

or greater than 0.4 is considered a positive result for the 

presence of PRRSV antibody and the assay has a sensitivity 

of 97.4% and specificity of 99.6%. Under experimental 

conditions the test is essentially 100% specific and 100% 

sensitive when testing serum from acutely infected swine. 

Chronically-infected swine have declining PRRSV antibody 

titers that are reflected with a decreasing S/P ratio that may 

fall below 0.4 within a few months of infection. In these 

situations (S/P < 0.4) it is easy to consider the serum sample 

positive since the history of the animal is known. In general, 

the range of the S/P ratios for the "false-negatives" is about 

0.2 to 0.4. False-positives with this assay under field 

conditions may run up to 2 to 3% and most of the S/P values 

are around 0.4 although higher values have been observed 

on occasion. In our laboratory about 98% of the negative 

animals tested (mostly pigs) have had a S/P value less than 

0.1 (mostly 0.00) and the remaining negative animals had S/P 

values ranging from 0.1 to less than 0.4. 

VACCINOLOGY 

Perhaps the best way to start this section is to answer the 

question, Why vaccinate? Well, one would vaccinate against 

a pathogen to prevent or attenuate the losses associated with 

a future infection of that pathogen. This is a simple concept 

until the pathogen is PRRSV, then the use of PRRSV vaccine 

becomes a complex issue. In the case of PRRS, one could 

hypothesize that it would be difficult to develop a traditional 

vaccine for this disease that would be efficacious. The 

reason for this statement is that under natural conditions, 

PRRSV-infected swine do not rapidly develop a protective 

immune response that clears the virus from their body, at 

least when compared to other swine viruses like swine 

influenza virus or porcine parvovirus. It appears that it may 

take several months for a normal pig's immune system to 

clear a wild-type PRRSV infection from its body. One could 

surmise that a vaccine would not stimulate any stronger of an 

immune response than wild-type virus. In fact, the vaccineinduced 

immune response might even be "weaker" than a 

wild-type virus immune response. Current commercially 

available PRRSV vaccines are produced as modified-live 

virus vaccine (MLV) or as inactivated or killed whole-virus 

vaccine (KV). In some countries (at least in the United 

States) autogenous KV may be commercially available. 

These are vaccines prepared specifically for a single herd 

from PRRSV isolated from that herd. 

Attenuated vaccine or MLV 

Susceptibility - Under experimental conditions, essentially all 

young and old swine will become infected with MLV when 

they are given a full dose according to vaccine label (23- 

25,37,43,54). Anecdotal field reports indicate that not all 

MLV-vaccinated swine will seroconvert. The incidence of this 

failure to seroconvert following vaccination is not clear, nor is 

it clear as to what might contribute to a failure of 

seroconversion. Understandably, improper handling or 

administration of vaccine virus could contribute to a failure of 

seroconversion, what other factors may play a role is 

unknown. As previously described, there is the lack of a


detectable humoral anamnestic response following 

homologous PRRSV challenge and this seems to be true 

following repeated injections of MLV. Perhaps this 

phenomenon is part of what is being reported as failure of 

seroconversion in the field. Another possibility is that PRRSV 

can be attenuated by repetitive passage in cell culture to such 

an extent that the virus may not even be infectious for pigs, 

even when given intramuscularly at a dose of 2 x 10 6 CCID50 

PRRSV (38). Interestingly, highly attenuated PRRSV may 

not even readily infect fetuses following direct inoculation 

(34). The mechanism behind the attenuation of PRRSV is 

unknown. 

Viremia - Following intramuscular injection of MLV, swine will 

replicate the virus and produce a viremia that usually lasts 

less than 4 weeks in young swine and less than 2 weeks in 

old swine. 

Transmission - Under experimental conditions swine 

vaccinated with MLV develop few if any clinical signs. 

Vaccinated boars can shed vaccine virus in semen and the 

duration of seminal shedding seems to be shorter when 

compared to wild-type virus infection (16,43,54). Vaccination 

of pregnant naive swine can result in transplacental infection 

and congenitally-infected piglets that appear normal at birth 

(34). Based on limited studies, the incidence of 

transplacental infection appears to be lower for MLVvaccinated 

pregnant sows when compared to pregnant sows 

infected with wild-type PRRSV (37). 

There are field reports of vaccinated animals shedding 

vaccine virus to naïve contacts based on seroconversion of 

the contacts (55). The potential duration for MLV shedding is 

not known. There are also reports of vaccine virus 

transmission between herds where the vaccine virus has 

been linked to clinical disease recognized mostly as 

reproductive failure (9). In these cases the clinical disease is 

attributed to MLV that has been serially passaged in swine 

and the vaccine virus reverted to a virulent state. There are 

genetic differences between the MLV and the putative MLV 

field isolates (42,56), how the genetic changes contribute to a 

reversion to virulence is not known. The incidence and 

impact of fetal infection with vaccine virus in the field is 

unknown. 

Serology - Under experimental conditions vaccinated animals 

seroconvert (>0.4 S/P ratio) about 100% of the time using the 

IDEXX ELISA. Based on this serologic test, the humoral 

immune response (antibody) will decay or decrease over time 

with some animals being reported as seronegative (


contact with pigs from different sources, always a potential 

PRRS disaster. Because of these difficulties most 

veterinarians try to control PRRS problems in pigs by 

depopulating the chronically-infected sites and changing 

animal flow. 

In addition to changing animal flow, there is another important 

strategy for PRRS control and prevention, the isolation of 

replacement animals before their introduction into the 

breeding herd. The need for isolation is that the introduction 

of replacement animals is probably the biggest risk faster for 

importing PRRSV into a herd. Once the decision for isolation 

has been made, the debate then ensues, How long is long 

enough for isolation? Duration of isolation can be dependent 

on the PRRS status of the recipient herd and of the 

replacement animals. Essentially, the goal of isolating 

replacement animals is to test them for evidence of 

seroconversion prior to their introduction into the herd. If the 

source of the replacement animals is considered to be 

PRRSV negative, then a few weeks of isolation is adequate 

to test for evidence of seroconversion. 

In most countries where PRRS is endemic there is a high 

percentage of PRRSV-infected herds. This makes the 

movement of PRRSV-infected swine likely and complicates 

the concept of isolation for incoming animals. If the 

replacement pigs are from a PRRS-positive herd more time in 

isolation is needed since the purpose of isolation is to let the 

incoming animals recover from any infection and stop 

shedding the infectious agent prior to their introduction into 

the herd. As stated previously, in the case of PRRS it could 

take months for some animals to stop shedding PRRSV and 

it may not be practical to isolate replacement gilts for 

extended periods of time. One solution to this problem of 

long-term isolation is to purchase replacement gilts at the 

time of weaning. It is assumed that by the time the gilts are 

of breeding age they will have become infected with PRRSV 

(via horizontal transmission from penmates), cleared the 

virus, and will not be shedding virus when they enter the 

breeding herd. This time frame is adequate most of the time. 

However, there is probably an occasional time when some 

gilts introduced into the herd are actively shedding PRRSV. 

This could be due to the effects of colostrum. Colostrum from 

a seropositive sow can suppress the clinical effects of a 

cogenital/neonatal PRRSV infection and may inhibit the rate 

of virus transmission among a group of pigs. Instead of 

PRRSV transmitting rapidly through the group, it "smolders" 

resulting in a slow rate of transmission with some gilts actively 

shedding virus upon entry into the breeding herd. 

In an effort to get around the complications of managing 

active PRRSV-infections in the isolation barn, it has been 

stressed to purchase replacement gilts from PRRS-positive 

herds that are clinically normal or stable. In these herds the 

incidence of active PRRSV-shedding pigs is lower and the 

purchase of replacement gilts as weaners has less risk. 

Although this practice may have less risk, it is difficult for pork 

producers to make the investment for housing replacement 

gilts from the time of weaning until breeding age. Some pork 

producers have had good success with purchasing older gilts 

from PRRS-stable herds and maintaining them in an 8-weeklong 

isolation period. Prior to entry into the herd, the gilts 

are tested for PRRSV antibody and their PRRS status if 

followed through isolation. If the group of replacement gilts is 

deemed PRRS stable, then they are kept. Generally, this 

scheme works well when buying gilts; however, when trying 

to follow up on these herds to evaluate the success of the 

isolation strategies, many of the herds have experienced 

another PRRS epidemic within a couple of years and the herd 

may end up in the endemic PRRS cycle described earlier. 

33 

Unfortunately, there are so many variables in these cases 

that it is difficult to determine the source of the virus for the 

cyclical PRRS epidemics. For example, the current PRRS 

epidemic may be due to introduction of new virus or endemic 

virus that has found a subpopulation of susceptible pigs. 

When virus is recovered and analyzed, there are at least 

some genetic changes when compared to virus isolated from 

previous epidemics. If there is "substantial" genetic change 

between the viruses, then it is assumed this virus is new to 

the herd. 

A different problem exists where the replacement gilts are 

PRRS-negative and they need to be prepared for entry into a 

PRRS-positive sow herd. One way to address this issue is 

combining the practice of isolation with the practice of 

acclimitization, i.e., exposing incoming replacement animals 

to the recipient herd’s endemic pathogens. In regards to 

PRRS, this practice usually involves several strategies. One 

is the use of cull animals and/or the “feed-back” of certain 

materials to the replacement animals with the hope that these 

actions will infect them with PRRSV (20). A concern about 

this style of acclimatization is if it would consistently work 

since adult swine do not consistently shed virus for any length 

of time and PRRSV is readily inactivated outside of living 

tissue. 

Another acclimatization plan that can be consistent at 

infecting replacement gilts with PRRSV involves operating the 

isolation barn under a continuous animal flow where 

horizontal PRRSV transmission is maintained by the 

continual addition of naïve replacements. This concept has 

merit when the replacement animals are PRRS negative and 

they are going into a PRRS positive herd. However, this 

practice is analogous to trying to maintain a fire, sometimes 

the fire may go out and sometimes the fire might burn out of 

control. The brakes or controls for this strategy involve 

another isolation facility that is maintained on an All-In/All-Out 

pig flow. The plan is to let the replacement animals “cool 

down” at this second site before they enter the herd. This 

may be a critical point since in PRRS-positive sow herds that 

are stable there may be subpopulations of sows that have 

become susceptible to a PRRSV infection. This strategy of 

maintaining a continuous flow acclimatization may require 

monitoring the PRRS status of the replacement gilts to make 

sure that the virus is still circulating in the isolation barn. A 

more consistent and perhaps more dangerous version of the 

continuous flow strategy is to harvest serum from PRRSVinfected 

pigs and then inject this serum into replacement gilts 

to establish active infection (6). 

Another possibility for immunizing PRRS-negative 

replacement gilts would be the use of PRRSV vaccines. In 

this situation all replacement gilts are vaccinated at the same 

time upon entry into the isolation facility. As discussed 

above, precautions should be taken concerning transmission 

risks of the MLV virus. Again, the 8 week period of isolation 

is a commonly used time-frame that reduces the chance of 

transmitting vaccine virus and allows the vaccinated gilt time 

to develop an immune response that would confer some 

clinical protection. The extent of this protection is difficult to 

predict. 

Over the years there has been extensive use of MLV and KV 

in a variety of strategies all designed to control and prevent 

PRRS. I assume there are proponents and opponents for 

every vaccine strategy that has been devised. This diverse 

response to vaccine use points out 1) the difficulty in 

designing any universal control and prevention plan; 2) the 

frustrations of trying to reconcile research results (where 

PRRSV vaccines can be demonstrated to be efficacious) with


real world experiences where the use of vaccine may appear 

to have no benefit in preventing a PRRSV strain from causing 

severe losses; and 3) the significance of preventing the 

introduction of PRRSV into the herd. 

In general, biosecurity protocols can be good at keeping out 

infected swine. This risk of importing PRRSV with 

replacement animals can be at about zero when the only 

introduction of genetic material is semen. The risk of semen 

is dependent on how the boar stud operates. Boar studs, like 

sow herds, can follow extensive biosecurity protocols, yet it is 

still possible for them to become infected with PRRSV. 

These PRRSV infections are attributed to an indirect area 

spread of virus. The exact route of this virus transmission is 

not understood and could be one of several as previously 

described. This indirect area spread of virus is a vexing 

problem because it is difficult to control the unknown. 

Moreover, it is difficult to assess the risk/benefit ratio of the 

economic investments required to control potential avian, 

insect, and mammalian vectors. These unknowns also make 

it difficult to determine the risk/benefit of depopulating and 

repopulating a swine herd with PRRS-negative stock, 

perhaps the easiest way to gain a PRRS-negative status. 

Depopulating and repopulating a swine herd with PRRSnegative 

stock is an expensive, but relatively quick option to 

gain a PRRS-negative status. A less expensive, but longer 

option is to follow test and removal strategies designed to 

convert a PRRS-positive stable herd into a PRRS-negative 

status (17). These test and removal strategies focus on 

removing PRRSV-positive animals using several diagnostic 

methods to find the positive animals. Serology may be the 

most efficient tool since there is a high probability that the 

only animals harboring PRRSV in a PRRS-stable herd would 

be the seropositive animals. Perhaps the biggest 

consideration in converting a PRRS-positive herd into a 

PRRS-negative status is how to keep PRRSV out of the 

herd. After all, the virus got into the herd somehow, and if 

that route of virus transmission is not stopped, then one 

would expect the herd to become infected again. 

FUTURE 

Although there are many strategies to control and prevent 

PRRS, it is clear that none of them by itself or in combination 

seem to be the total answer to the PRRS problem. No matter 

how good the present biosecurity protocol may be for a herd, 

there is always the concern that a PRRS epidemic is just 

around the corner. If the PRRS problem is going to be solved, 

then something new will have to happen. 

At present, there are two "something new" pathways; one 

involves the eradication of PRRS, and the second involves 

the administration of some agent to swine that would be a 

significant improvement over current vaccines. Elimination of 

PRRSV from a farm can be achieved, but it will be difficult to 

implement regional and national PRRS eradication efforts 

without a better understanding of how to prevent indirect 

transmission of PRRSV, an apparent random event. 

Moreover, for the foreseeable future, the concentration of the 

swine industry may plague any eradication efforts. There are 

a number of research initiatives around the world 

investigating the second path. Scientists at these 

laboratories are applying cutting-edge technology towards 

developing better PRRSV vaccines. The direction of these 

research programs falls into two broad areas, adjuvants 

(cytokines [21,61] and immunoactive peptides [41]), and 

vaccines (DNA [47], PRRSV-deletion mutant [60], and viraland 

bacterial-vectored PRRSV genes [3,4]). 

34 

For "something new" to happen, the research community will 

need to be very clever in applying current technology with 

limited resources to gain the most information possible. 

Perhaps this meeting will provide a good forum on directing 

future PRRS research. 

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WORKSHOP 1 

PRRS 

Epidemiology 

37


38

porcine reproductive and respiratory syndrome

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