Risk factors for African swine fever incursion in Romanian domestic farms during 2019.

African swine fever (ASF) entered Georgia in 2007 and the EU in 2014. In the EU, the virus primarily spread in wild boar (Sus scrofa) in the period from 2014-2018. However, from the summer 2018, numerous domestic pig farms in Romania were affected by ASF. In contrast to the existing knowledge on ASF transmission routes, the understanding of risk factors and the importance of different transmission routes is still limited. In the period from May to September 2019, 655 Romanian pig farms were included in a matched case-control study investigating possible risk factors for ASF incursion in commercial and backyard pig farms. The results showed that close proximity to outbreaks in domestic farms was a risk factor in commercial as well as backyard farms. Furthermore, in backyard farms, herd size, wild boar abundance around the farm, number of domestic outbreaks within 2 km around farms, short distance to wild boar cases and visits of professionals working on farms were statistically significant risk factors. Additionally, growing crops around the farm, which could potentially attract wild boar, and feeding forage from ASF affected areas to the pigs were risk factors for ASF incursion in backyard farms.

Postweaning multisystemic wasting syndrome in Danish pig herds: productivity, clinical signs and pathology.

A case-control study of 74 herds with postweaning multisystemic wasting syndrome (pmws) and 74 matched control herds was carried out. In the case herds the mortality rates of weaner and finisher pigs were 11.2 and 5.2 per cent respectively, compared with 3.1 and 3.2 per cent in the control herds. In most case herds, pmws developed within the first four weeks after weaning. Wasting, diarrhoea and respiratory signs were observed in 10 per cent of the weaner pigs (7 to 30 kg) in the case herds compared with 7 per cent in the control herds. The average daily gains of the weaner pigs and finisher pigs were 36 g and 52 g less in the case herds than in the control herds. By examining three weaner pigs from each herd the pmws diagnosis was confirmed by histopathology and immunohistochemistry in 78 per cent of the case herds, but at least one pmws-positive weaner pig was found in 19 of the control herds. The prevalence of pmws-positive pigs among illthriven weaner pigs was 45 per cent (101/222) in the case herds, and 12 per cent (27/222) in the control herds. Specific gross pathological findings were associated with a positive pmws diagnosis; pigs with heavy, rubber-like lungs, atonic intestines, and enlarged bronchial and inguinal lymph nodes, had a 0.7 probability of a positive pmws diagnosis by laboratory examinations. However, for illthriven pigs, this probability of having pmws was equal in the case herds and the control herds.

Short time window for transmissibility of African swine fever virus from a contaminated environment.

Since the introduction of African swine fever virus (ASFV) into the Baltic states and Poland in 2014, the disease has continued to spread within these regions. In 2017, the virus spread further west and the first cases of disease were reported in the Czech Republic and Romania, in wild boar and domestic pigs, respectively. To control further spread, knowledge of different modes of transmission, including indirect transmission via a contaminated environment, is crucial. Up until now, such an indirect mode of transmission has not been demonstrated. In this study, transmission via an environment contaminated with excretions from ASFV-infected pigs was investigated. Following euthanasia of pigs that were infected with an isolate of ASFV from Poland (POL/2015/Podlaskie/Lindholm), healthy pigs were introduced into the pens, in which the ASFV-infected pigs had been housed. Introduction was performed at 1, 3, 5 or 7 days, following euthanasia of the infected pig groups. Pigs, that were introduced into the contaminated environment after 1 day, developed clinical disease within 1 week, and both ASFV DNA and infectious virus were isolated from their blood. However, pigs introduced into the contaminated pens after 3, 5 or 7 days did not develop any signs of ASFV infection and no viral DNA was detected in blood samples obtained from these pigs within the following 3 weeks. Thus, it was shown that exposure of pigs to an environment contaminated with ASFV can result in infection. However, the time window for transmissibility of ASFV seems very limited, and, within our experimental system, there appears to be a rapid decrease in the infectivity of ASFV in the environment.

Efficacy and safety of simultaneous vaccination with two modified live virus vaccines against porcine reproductive and respiratory syndrome virus types 1 and 2 in pigs.

The objective of the study was to compare responses of pigs vaccinated with a PRRS MLV vaccine against PRRSV-1 or PRRSV-2 with the responses of pigs vaccinated simultaneously with both vaccines. Furthermore, the efficacy of the two PRRSV MLV vaccination strategies was assessed following challenge. The experimental design included four groups of 4-weeks old SPF-pigs. On day 0 (DPV0), groups 1-3 (N=18 per group) were vaccinated with modified live virus vaccines (MLV) containing PRRSV-1 virus (VAC-T1), PRRSV-2 virus (VAC-T2) or both (VAC-T1T2). One group was left unvaccinated (N=12). On DPV 62, the pigs from groups 1-4 were mingled in new groups and challenged (DPC 0) with PRRSV-1, subtype 1, PRRSV-1, subtype 2 or PRRSV-2. On DPC 13/14 all pigs were necropsied. Samples were collected after vaccination and challenge. PRRSV was detected in all vaccinated pigs and the majority of the pigs were positive until DPV 28, but few of the pigs were still viremic 62 days after vaccination. Virus was detected in nasal swabs until DPV 7-14. No overt clinical signs were observed after challenge. PRRSV-2 vaccination resulted in a clear reduction in viral load in serum after PRRSV-2 challenge, whereas there was limited effect on the viral load in serum following challenge with the PRRSV-1 strains. Vaccination against PRRSV-1 had less impact on viremia following challenge. The protective effects of simultaneous vaccination with PRRSV Type 1 and 2 MLV vaccines and single PRRS MLV vaccination were comparable. None of the vaccines decreased the viral load in the lungs at necropsy. In conclusion, simultaneous vaccination with MLV vaccines containing PRRSV-1 and PRRSV-2 elicited responses comparable to single vaccination and the commercial PRRSV vaccines protected only partially against challenge with heterologous strains. Thus, simultaneous administration of the two vaccines is an option in herds with both PRRSV types.

Experimental Infection of Young Pigs with an Early European Strain of Porcine Epidemic Diarrhoea Virus and a Recent US Strain.

Outbreaks of porcine epidemic diarrhoea (PED) were reported across Europe during the 1980s and 1990s, but only sporadic outbreaks occurred in recent years. PED virus (PEDV) spread for the first time into the USA in 2013 and has caused severe economic losses. Retrospectively, it was found that two different strains of PEDV have been introduced into the United States, both are closely related to strains circulating in China where a new wave of the disease occurred from 2010 onwards. Since autumn 2014, new outbreaks of PED have occurred in Europe. In this study, weaned piglets were inoculated with an early European isolate (Br1/87) or faecal/intestinal suspensions derived from pigs infected with a recent European strain of PEDV (from Germany) or a US strain of PEDV. No evidence for infection resulted from inoculation of pigs with the German sample that contained high levels of PEDV RNA; there were no clinical signs, excretion of viral RNA or anti-PEDV antibody production. In contrast, all the pigs in the other two groups showed evidence of infection. Mild clinical signs of disease, mainly diarrhoea, occurred in piglets inoculated with the Br1/87 and US PEDV strains. PEDV RNA was detected throughout the intestine in euthanized animals at 4 days post-inoculation. In addition, within these animals, low levels of viral RNA were detected in lungs and livers with higher levels in spleens. Seroconversion against PEDV occurred in all surviving infected animals within 10 days. PEDV RNA excretion occurred for at least 2 weeks. The US PEDV RNA was detected at low levels in serum samples on multiple days. It is apparent that current diagnostic systems can detect infection by the different virus strains.

Genetic and biological characterization of a Porcine Reproductive and Respiratory Syndrome Virus 2 (PRRSV-2) causing significant clinical disease in the field.

Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is the cause of severe reproductive and respiratory disease in swine worldwide. In Denmark, both PRRSV-1 and PRRSV-2 are circulating and approximately 35% of pig herds are seropositive for PRRSV. In November 2010, a pig herd in the Northern part of Denmark experienced an infection with PRRSV-2 with clinical signs that were much more severe than normally reported from current Danish PRRSV-2 affected herds. Due to the clinical observations of reproductive failure in sows and high mortality in piglets, it was speculated that a new, more pathogenic or vaccine evading PRRSV strain had emerged in Denmark. The overall aim of the present study was to perform a genetic and biological characterization of the virus isolated from the diseased herd. Complete genome sequencing of isolates from this herd revealed that although the case strain had some unique genetic features including a deduced 3 amino acid deletion, it was in overall very similar to the other PRRS-2 viruses circulating in Denmark. In an experimental trial in growing pigs, no overt clinical signs or pathology were observed following intranasal inoculation with the new virus isolate. Virus shedding, acute phase protein responses and serological responses were comparable to those seen after experimental challenge with a Danish PRRSV-2 reference strain isolated in 1997. Vaccination with a commercial modified live PRRSV-2 vaccine had a clear reducing effect on virus shedding, magnitude, and duration of viremia and viral load in the lungs. Overall, the results indicate that the severe disease observed in the field was contributed by additional factors in combination with the PRRS virus infection.

Urgent request on avian influenza.

Highly pathogenic avian influenza (HPAI) H5N8 is currently causing an epizootic in Europe, infecting many poultry holdings as well as captive and wild bird species in more than 10 countries. Given the clear clinical manifestation, passive surveillance is considered the most effective means of detecting infected wild and domestic birds. Testing samples from new species and non-previously reported areas is key to determine the geographic spread of HPAIV H5N8 2016 in wild birds. Testing limited numbers of dead wild birds in previously reported areas is useful when it is relevant to know whether the virus is still present in the area or not, e.g. before restrictive measures in poultry are to be lifted. To prevent introduction of HPAIV from wild birds into poultry, strict biosecurity implemented and maintained by the poultry farmers is the most important measure. Providing holding-specific biosecurity guidance is strongly recommended as it is expected to have a high impact on the achieved biosecurity level of the holding. This is preferably done during peace time to increase preparedness for future outbreaks. The location and size of control and in particular monitoring areas for poultry associated with positive wild bird findings are best based on knowledge of the wider habitat and flight distance of the affected wild bird species. It is recommended to increase awareness among poultry farmers in these established areas in order to enhance passive surveillance and to implement enhanced biosecurity measures including poultry confinement. There is no scientific evidence suggesting a different effectiveness of the protection measures on the introduction into poultry holdings and subsequent spread of HPAIV when applied to H5N8, H5N1 or other notifiable HPAI viruses.

Characterization of a Novel Chimeric Swine Enteric Coronavirus from Diseased Pigs in Central Eastern Europe in 2016.

During a severe outbreak of diarrhoea and vomiting in a pig herd in Central Eastern Europe, faecal samples were tested positive for porcine epidemic diarrhoea virus (PEDV) and negative for transmissible gastroenteritis virus (TGEV) using a commercial RT-qPCR assay that can detect both of these coronaviruses. However, further analyses, using other TGEV- and PEDV-specific RT-qPCR assays, provided results inconsistent with infection by either of these viruses. Sequencing of an amplicon (ca. 1.6 kb), generated by an RT-PCR specific for the PEDV S-gene, indicated a very close similarity (ca. 99% identity) to recently described chimeric viruses termed swine enteric coronaviruses (SeCoVs). These viruses (with an RNA genome of ca. 28 kb) were first identified in Italy in samples from 2009 but have not been detected there since 2012. A closely related virus was detected in archived samples in Germany from 2012, but has not been detected subsequently. Building on the initial sequence data, further amplicons were generated and over 9 kb of sequence corresponding to the 3'-terminus of the new SeCoV genome was determined. Sequence comparisons showed that the three known SeCoVs are ≥98% identical across this region and contain the S-gene and 3a sequences from PEDV within a backbone of TGEV, but the viruses are clearly distinct from each other. It is demonstrated, for the first time, that pigs from within the SeCoV-infected herd seroconverted against PEDV but tested negative in a TGEV-specific ELISA that detects antibodies against the S protein. These results indicate that SeCoV is continuing to circulate in Europe and suggest it can cause a disease that is very similar to PED. Specific detection of the chimeric SeCoVs either requires development of a new diagnostic RT-qPCR assay or the combined use of assays targeting the PEDV S-gene and another part of the TGEV genome.

Assessing the potential spread and maintenance of foot-and-mouth disease virus infection in wild ungulates: general principles and application to a specific scenario in Thrace.

Foot-and-mouth disease (FMD), due to infection with serotype O virus, occurred in wild boar and within eleven outbreaks in domestic livestock in the south-east of Bulgaria, Thrace region, in 2011. Hence, the issue of the potential for the spread and maintenance of FMD virus (FMDV) infection in a population of wild ungulates became important. This assessment focused on the spread and maintenance of FMDV infection within a hypothetical wild boar and deer population in an environment, which is characterized by a climate transitional between Mediterranean and continental and variable wildlife population densities. The assessment was based on three aspects: (i) a systematic review of the literature focusing on experimental infection studies to identify the parameters describing the duration of FMDV infection in deer and wild boar, as well as observational studies assessing the occurrence of FMDV infection in wild deer and wild boar populations, (ii) prevalence survey data of wild boar and deer in Bulgaria and Turkey and (iii) an epidemiological model, simulating the host-to-host spread of FMDV infections. It is concluded, based on all three aspects, that the wildlife population in Thrace, and so wildlife populations in similar ecological settings, are probably not able to maintain FMD in the long term in the absence of FMDV infection in the domestic host population. However, limited spread of FMDV infection in time and space in the wildlife populations can occur. If there is a continued cross-over of FMDV between domestic and wildlife populations or a higher population density, virus circulation may be prolonged.

Experimental reproduction of postweaning multisystemic wasting syndrome (PMWS) in pigs in Sweden and Denmark with a Swedish isolate of porcine circovirus type 2.

An experimental model using 3-day-old snatch-farrowed colostrum-deprived piglets co-infected with porcine circovirus type 2 (PCV2) and porcine parvovirus (PPV) is at present one of the best methods to study factors affecting development of postweaning multisystemic wasting syndrome (PMWS). A Swedish isolate of PCV2 (S-PCV2) retrieved in 1993 from a healthy pig has been used in this model to reproduce PMWS in pigs from Northern Ireland. This virus has been present in the Swedish pig population for at least a decade without causing any known PMWS disease problems, despite its potential pathogenicity. The reasons for this are unknown, but could be related to genetics, absence of triggers for PCV2 upregulation (infectious agent and/or management forms) within Swedish pig husbandry. In order to confirm the pathogenicity of S-PCV2, Swedish and Danish pigs were experimentally infected with this isolate according to the established model. Swedish pigs were also infected with a reference isolate of PCV2 (PCV2-1010) to compare the severity of disease caused by the two isolates in Swedish pigs. Both Danish and Swedish pigs developed PMWS after the experimental infection with S-PCV2. Antibodies to PCV2 developed later and reached lower levels in serum from pigs infected with S-PCV2 than in pigs inoculated with PCV2-1010. In general, pigs infected with S-PCV2 showed more severe clinical signs of disease than pigs infected with PCV2-1010, but pigs from all PCV2-inoculated groups displayed gross and histological lesions consistent with PMWS. All pigs inoculated with PPV, alone or in combination with PCV2, displayed interleukin-10 responses in serum while only pigs infected with PPV in combination with PCV2 showed interferon-alpha in serum on repeated occasions. Thus, the pathogenicity of S-PCV2 was confirmed and a role for cytokines in the etiology of PMWS was indicated.

Mapping the antigenic structure of porcine parvovirus at the level of peptides.

The antigenic structure of the capsid proteins of porcine parvovirus (PPV) was investigated. A total of nine linear epitopes were identified by Pepscan using porcine or rabbit anti-PPV antisera. No sites were identified with a panel of neutralising monoclonal antibodies (MAbs). All epitopes were located in the region corresponding to the major capsid protein VP2. Based on this information, and on analogy to other autonomous parvoviruses, 24 different peptides were synthesised, coupled to keyhole limpet haemocyanin (KLH) and used to immunise rabbits. Most antisera were able to bind viral protein. Only peptides from the N-terminal part of VP2 were able to induce virus-neutralising antibodies, although at low levels. A similar neutralising activity could be obtained in pigs. The exposure of the N-terminus was shown in full virions, both by immunoelectron microscopy and absorption experiments. It is concluded that in PPV, the VP2 N-terminus is involved in virus neutralisation (VN) and peptides from this region are therefore primary targets for developing peptide-based vaccines against this virus.

Cytokine mRNA profiles in bronchoalveolar cells of piglets experimentally infected in utero with porcine reproductive and respiratory syndrome virus: association of sustained expression of IFN-gamma and IL-10 after viral clearance.

An experimental model was used to investigate mRNA cytokine profiles in bronchoalvolar cells (BALC) from piglets, infected in utero with porcine reproductive and respiratory syndrome virus (PRRSV). The BALC's were analyzed for the cytokines TNF-alpha, IFN-gamma, IL-8, IL-10, and IL-12(p40) by real-time TaqMan polymerase chain reaction in 2-, 4-, and 6-week-old piglets, respectively. High levels of IFN-gamma mRNA was detected in all piglets, while IL-10 was upregulated in 2-week-old piglets, was at normal levels in 4-week-old piglets, and elevated again in 6-week-old piglets. IL-12 was weakly elevated in all three age groups. Virus was reduced by 50% in 4-week-old piglets and cleared by 6 weeks of age. The sustained expression of IFNgamma and reduction of IL-10 production indicate an important role for these cytokines in immunity to PRRSV.

Diagnosis of PRRS.

This paper reviews various diagnostic methods for the detection of porcine reproductive and respiratory syndrome (PRRS) virus or antibodies to PRRS virus reported during the period from 1991 to 1995. In addition, experience from a European Community Concerted Action and especially Danish experiences concerning serological tests are presented. It is concluded that, in general, serological diagnosis with a high specificity and sensitivity is easy to perform on a herd level. However, no serological test has proven to be suitable for individual animal certification.

Survival of Aujeszky's disease virus in slurry at various temperatures.

Survival of Aujeszky's disease virus in pig slurry was investigated during anaerobic storage at 5, 20, 35, 40, 45, 50 and 55 degrees C using 100-ml laboratory models simulating the conditions in slurry tanks during winter and summer seasons and during anaerobic digestion in batch reactors. The inactivation rate was found to increase with increasing temperature. Virus was inactivated at 5 and 20 degrees C in 15 weeks and 2 weeks, respectively. At 35 degrees C (mesophilic conditions) the virus was inactivated in 5 hours and at 55 degrees C (thermophilic conditions) no virus could be detected after 10 minutes.

Hematological and immunological parameters of 4 1/2-month old pigs infected with PRRS virus.

4 1/2-month old SPF pigs were experimentally infected with PRRS virus. Blood samples were collected with regular intervals up to day 35 post inoculation (PI). Serum was used for PRRS virus isolation and antibody detection and stabilized blood for total leucocyte counts, differential counts and characterization of lymphocyte subpopulations by flow cytometry analysis using monoclonal antibodies specific for porcine CD2, CD4 and CD8. After an initial viremic period of 1-7 days duration for individual pigs, PRRS virus was intermittently detected in pigs up to day 18 PI. All pigs had developed antibodies against PRRS virus by day 14 PI. Total blood leucocyte counts and lymphocyte counts were significantly decreased for a few days shortly after infection, but had returned to pre-infection levels on day 8-10 PI. A major change in the distribution of lymphocyte subpopulations was observed on day 3 PI, where the percentages of CD2+, CD4+ and CD8+ cells were significantly decreased. However, the percentages of these lymphocyte subsets quickly returned to approximately pre-infection values. The observed changes of the parameters examined do not indicate long-term systemic immunosuppression of the infected pigs.

Isolation of porcine reproductive and respiratory syndrome (PRRS) virus in a Danish swine herd and experimental infection of pregnant gilts with the virus.

The first case of porcine reproductive and respiratory syndrome (PRRS) in Denmark was diagnosed in March 1992 by the detection of specific antibodies against PRRS virus in serum samples originating from sows in a herd located on the island of Als. Subsequently, PRRS virus was isolated from a 200-sow farrow-to-finish herd with clinical signs consistent with PRRS. The virus was isolated by inoculation of pleural fluid from a stillborn piglet onto porcine pulmonary alveolar macrophages. The isolate was identified as PRRS virus by staining with a specific antiserum. By electron microscopy, the virus particle was found to be spherical and enveloped, measuring 45-55 nm in diameter and containing a 30-35 nm nucleocapsid. Only minor antigenic differences were found between the Danish and a Dutch isolate. Following intranasal inoculation of 3 pregnant gilts with the Danish isolate transplacental infection was demonstrated by the re-isolation of PRRS virus from approximately 45% of the piglets from the experimentally infected gilts. However, the experimental infection produced no significant reproductive disorders or other clinical signs. At autopsy, histopathological examination revealed slight interstitial pneumonia in a few piglets.

Semen from boars infected with porcine reproductive and respiratory syndrome virus (PRRSV) contains antibodies against structural as well as nonstructural viral proteins.

The seminal excretion of antibodies against porcine reproductive and respiratory syndrome virus (PRRSV) was examined in a group of five boars experimentally infected by the nasopharyngeal route. By using phage-displayed peptide epitopes from the PRRSV replicase and envelope glycoproteins as ELISA antigen, we were able to separately and specifically assay antibody responses against structural and nonstructural viral proteins. Antibodies against structural as well as nonstructural viral proteins were consistently found in the semen of all boars, beginning from 1-4 weeks postinfection. This is the first report documenting the presence of anti-PRRSV antibodies in boar semen. Seminal antiviral IgA was also detected, and we observed a correlation between seminal IgA responses against nonstructural viral proteins, and the duration of PRRSV RNA excretion in semen. The implications of these findings for the diagnostics and pathogenesis of venereal PRRSV infection are discussed.

A case-control questionnaire survey of risk factors for porcine reproductive and respiratory syndrome (PRRS) seropositivity in Danish swine herds.

Sixty-eight case herds seropositive to porcine reproductive and respiratory syndrome (PRRS) were compared to 128 seronegative controls in a double-blinded questionnaire survey. The study indicated no increased risk of PRRS seropositivity for herds using artificial insemination with semen from PRRS seropositive AI-stations. Also the herd-size was non-related to the risk of PRRS seropositivity, indicating that air-borne spread of PRRS may not have been a predominant feature in Denmark. Introduction of replacement breeding animals from seropostive breeding- and multiplying herds significantly increased the risk of a herd being PRRS seropositive, as did introduction of 25 kg pigs for feeding. PRRS seropositivity was in the farmers' opinions associated with abortions in sows, early farrowing, high postweaning mortality and low weight gain in fattening pigs. However, the reported frequencies of probelms were relatively low.

Heterologous challenge with porcine reproductive and respiratory syndrome (PRRS) vaccine virus: no evidence of reactivation of previous European-type PRRS virus infection.

In Denmark, a porcine reproductive and respiratory syndrome virus (PRRSV) control programme, comprising vaccination of seropositive herds with a live American type PRRSV vaccine, was started in 1996. In several of these herds, spread of vaccine virus from vaccinated 3-18 week old pigs to non-vaccinated sows was demonstrated by the isolation of vaccine virus from fetuses and stillborn piglets. Surprisingly, sows infected with the American type vaccine strain consistently exhibited significantly stronger serological responses towards European type PRRSV than American type PRRSV. In order to elucidate whether the unexpectedly strong serological reaction towards European-type PRRSV in American type PRRSV infected sows was due to a booster reaction, or reactivation of an unrecognized, latent infection in the sows with European type PRRSV, a challenge study with the vaccine was carried out. In this study, the stronger serological response towards European type PRRSV than towards American type PRRSV was reproduced, and reactivation of the previous natural infection with European PRRSV could neither be demonstrated by virus isolation nor by RT-PCR. So, the increase in antibody titers towards European PRRSV in previously European PRRSV infected pigs after challenge with the vaccine strain seems to be the result of a boosting effect on the immune system, induced by the heterologous vaccine PRRSV strain.

Sensitive detection and typing of porcine reproductive and respiratory syndrome virus by RT-PCR amplification of whole viral genes.

Following the recent use of a live vaccine against porcine reproductive and respiratory syndrome virus (PRRSV) in Denmark, both American (vaccine) and European-type PRRSV now coexist in Danish herds. This situation highlighted a requirement for supplementary tests for precise virus-typing. As a result, we developed a RT-PCR assay able to detect as well as type PRRSV. To provide maximal sequence information, complete viral open reading frames (ORFs 5 and 7) were targeted for amplification. The RT-PCR test was able to amplify complete PRRSV ORFs from complex materials such as boar semen containing as little as 1 TCID50 ml(-1) of PRRSV. Typing of viruses was accomplished by any one of three strategies: (i) use of type-specific PCR primers, (ii) size determination of ORF 7 amplicons, (iii) DNA sequencing. All three typing strategies showed complete concordance with the currently used method of typing with monoclonal antibodies (MAbs) when used on a panel of PRRSV field isolates covering the period 1992-1997. The ORF 7-based test had particularly desirable characteristics, namely, highly sensitive detection of PRRSV without apparent type bias, typing of the detected virus, discrimination between pure and mixed virus populations, and semi-quantitative assessment of type ratios in mixed populations, all in a single PCR reaction. In addition, the obtained sequence data were used to predict two simple and rapid strategies (single-enzyme restriction length polymorphy analysis and oligonucleotide hybridization) for confirmation of the specificity of ORF 7 RT-PCR reactions. As such, the RT-PCR assay provides a new, powerful diagnostic tool to study the population dynamics between present and emerging PRRSV-types.