Serological evidence for the presence of wobbly possum disease virus in Australia.

Wobbly possum disease virus (WPDV) is an arterivirus that was originally identified in common brushtail possums (Trichosurus vulpecula) in New Zealand, where it causes severe neurological disease. In this study, serum samples (n = 188) from Australian common brushtail, mountain brushtail (Trichosurus cunninghami) and common ringtail (Pseudocheirus peregrinus) possums were tested for antibodies to WPDV using ELISA. Antibodies to WPDV were detected in possums from all three species that were sampled in the states of Victoria and South Australia. Overall, 16% (30/188; 95% CI 11.0-22.0) of possums were seropositive for WPDV and 11.7% (22/188; 95% CI 7.5-17.2) were equivocal. The frequency of WPDV antibody detection was the highest in possums from the two brushtail species. This is the first reported serological evidence of infection with WPDV, or an antigenically similar virus, in Australian possums, and the first study to find antibodies in species other than common brushtail possums. Attempts to detect viral RNA in spleens by PCR were unsuccessful. Further research is needed to characterise the virus in Australian possums and to determine its impact on the ecology of Australian marsupials.

Clinical Characterization of Host Response to Simian Hemorrhagic Fever Virus Infection in Permissive and Refractory Hosts: A Model for Determining Mechanisms of VHF Pathogenesis.

Simian hemorrhagic fever virus (SHFV) causes a fulminant and typically lethal viral hemorrhagic fever (VHF) in macaques (Cercopithecinae: Macaca spp.) but causes subclinical infections in patas monkeys (Cercopithecinae: Erythrocebus patas). This difference in disease course offers a unique opportunity to compare host responses to infection by a VHF-causing virus in biologically similar susceptible and refractory animals. Patas and rhesus monkeys were inoculated side-by-side with SHFV. Unlike the severe disease observed in rhesus monkeys, patas monkeys developed a limited clinical disease characterized by changes in complete blood counts, serum chemistries, and development of lymphadenopathy. Viral RNA was measurable in circulating blood 2 days after exposure, and its duration varied by species. Infectious virus was detected in terminal tissues of both patas and rhesus monkeys. Varying degrees of overlap in changes in serum concentrations of interferon (IFN)-γ, monocyte chemoattractant protein (MCP)-1, and interleukin (IL)-6 were observed between patas and rhesus monkeys, suggesting the presence of common and species-specific cytokine responses to infection. Similarly, quantitative immunohistochemistry of livers from terminal monkeys and whole blood flow cytometry revealed varying degrees of overlap in changes in macrophages, natural killer cells, and T-cells. The unexpected degree of overlap in host response suggests that relatively small subsets of a host's response to infection may be responsible for driving hemorrhagic fever pathogenesis. Furthermore, comparative SHFV infection in patas and rhesus monkeys offers an experimental model to characterize host⁻response mechanisms associated with viral hemorrhagic fever and evaluate pan-viral hemorrhagic fever countermeasures.

Antiviral Innate Immune Response Interferes with the Formation of Replication-Associated Membrane Structures Induced by a Positive-Strand RNA Virus.

Infection with nidoviruses like corona- and arteriviruses induces a reticulovesicular network of interconnected endoplasmic reticulum (ER)-derived double-membrane vesicles (DMVs) and other membrane structures. This network is thought to accommodate the viral replication machinery and protect it from innate immune detection. We hypothesized that the innate immune response has tools to counteract the formation of these virus-induced replication organelles in order to inhibit virus replication. Here we have investigated the effect of type I interferon (IFN) treatment on the formation of arterivirus-induced membrane structures. Our approach involved ectopic expression of arterivirus nonstructural proteins nsp2 and nsp3, which induce DMV formation in the absence of other viral triggers of the interferon response, such as replicating viral RNA. Thus, this setup can be used to identify immune effectors that specifically target the (formation of) virus-induced membrane structures. Using large-scale electron microscopy mosaic maps, we found that IFN-ß treatment significantly reduced the formation of the membrane structures. Strikingly, we also observed abundant stretches of double-membrane sheets (a proposed intermediate of DMV formation) in IFN-ß-treated samples, suggesting the disruption of DMV biogenesis. Three interferon-stimulated gene products, two of which have been reported to target the hepatitis C virus replication structures, were tested for their possible involvement, but none of them affected membrane structure formation. Our study reveals the existence of a previously unknown innate immune mechanism that antagonizes the viral hijacking of host membranes. It also provides a solid basis for further research into the poorly understood interactions between the innate immune system and virus-induced replication structures. IMPORTANCE: Viruses with a positive-strand RNA genome establish a membrane-associated replication organelle by hijacking and remodeling intracellular host membranes, a process deemed essential for their efficient replication. It is unknown whether the cellular innate immune system can detect and/or inhibit the formation of these membrane structures, which could be an effective mechanism to delay viral RNA replication. In this study, using an expression system that closely mimics the formation of arterivirus replication structures, we show for the first time that IFN-ß treatment clearly reduces the amount of induced membrane structures. Moreover, drastic morphological changes were observed among the remaining structures, suggesting that their biogenesis was impaired. Follow-up experiments suggested that host cells contain a hitherto unknown innate antiviral mechanism, which targets this common feature of positive-strand RNA virus replication. Our study provides a strong basis for further research into the interaction of the innate immune system with membranous viral replication organelles.

The Cholera Toxin B Subunit (CTB) Fused to the Porcine Arterivirus Matrix M and GP5 Envelope Proteins Fails to Enhance the GP5-Specific Antibody Response in Pigs Immunized with Adenovectors.

The porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus of the Arteriviridae family. As the current commercial vaccines are incompletely protective effective against PRRSV infection, we developed a vaccine strategy using replicating but non-disseminating adenovectors (rAdVs) expressing the PRRSV M matrix protein in fusion with the neutralizing major epitope-carrying GP5 envelope protein (Roques et al. in Vet Res 44:17, 2013). Although production of GP5-specific antibodies (Abs) was observed, no PRRSV-specific neutralizing Abs (NAbs) were induced in pigs given the rAdVs expressing M-GP5 or M-GP5m (GP5m being a mutant form of GP5). Nevertheless, partial protection was observed in the M-GP5m-rAdV-inoculated pigs experimentally infected with PRRSV. Here, we determined the impact of the cholera toxin B subunit (CTB, known for its adjuvant effect) in fusion with the C-terminus of M-GP5m on the Ab response to PRRSV. Three-week-old pigs were immunized twice both intramuscularly and intranasally at 3-week intervals with rAdV-expressing the green fluorescent protein (rAdV-GFP), rAdV-M-GP5m, or rAdV-M-GP5m-CTB. Pigs immunized with rAdV-M-GP5m showed a high level of serum GP5-specific Abs (as determined by an indirect ELISA). In contrast, CTB in fusion with M-GP5m had an unexpected severe negative impact on GP5-specific Ab production. PRRSV-specific NAbs could not be detected in any pigs of all groups.

Modulation of innate immune signaling by nonstructural protein 1 (nsp1) in the family Arteriviridae.

Arteriviruses infect immune cells and may cause persistence in infected hosts. Inefficient induction of pro-inflammatory cytokines and type I IFNs are observed during infection of this group of viruses, suggesting that they may have evolved to escape the host immune surveillance for efficient survival. Recent studies have identified viral proteins regulating the innate immune signaling, and among these, nsp1 (nonstructural protein 1) is the most potent IFN antagonist. For porcine reproductive and respiratory syndrome virus (PRRSV), individual subunits (nsp1α and nsp1ß) of nsp1 suppress type I IFN production. In particular, PRRSV-nsp1α degrades CREB (cyclic AMP responsive element binding)-binding protein (CBP), a key component of the IFN enhanceosome, whereas PRRSV-nsp1ß degrades karyopherin-α1 which is known to mediate the nuclear import of ISGF3 (interferon-stimulated gene factor 3). All individual subunits of nsp1 of PRRSV, equine arteritis virus (EAV), lactate dehydrogenase-elevating virus (LDV), and simian hemorrhagic fever virus (SHFV) appear to contain IFN suppressive activities. As with PRRSV-nsp1α, CBP degradation is evident by LDV-nsp1α and partly by SHFV-nsp1γ. This review summarizes the biogenesis and the role of individual subunits of nsp1 of arteriviruses for innate immune modulation.

Regulatory T cells in arterivirus and coronavirus infections: do they protect against disease or enhance it?

Regulatory T cells (T(regs)) are a subset of T cells that are responsible for maintaining peripheral immune tolerance and homeostasis. The hallmark of T(regs) is the expression of the forkhead box P3 (FoxP3) transcription factor. Natural regulatory T cells (nT(regs)) are a distinct population of T cells that express CD4 and FoxP3. nTregs develop in the thymus and function in maintaining peripheral immune tolerance. Other CD4(+), CD4(-)CD8(-), and CD8(+)CD28(-) T cells can be induced to acquire regulatory function by antigenic stimulation, depending on the cytokine milieu. Inducible (or adaptive) T(regs) frequently express high levels of the interleukin 2 receptor (CD25). Atypical T(regs) express FoxP3 and CD4 but have no surface expression of CD25. Type 1 regulatory T cells (Tr1 cells) produce IL-10, while T helper 3 cells (Th3) produce TGF-ß. The function of inducible T(regs) is presumably to maintain immune homeostasis, especially in the context of chronic inflammation or infection. Induction of T(regs) in coronaviral infections protects against the more severe forms of the disease attributable to the host response. However, arteriviruses have exploited these T cell subsets as a means to dampen the immune response allowing for viral persistence. T(reg) induction or activation in the pathogenesis of disease has been described in both porcine reproductive and respiratory syndrome virus, lactate dehydrogenase elevating virus, and mouse hepatitis virus. This review discusses the development and biology of regulatory T cells in the context of arteriviral and coronaviral infection.

Simian hemorrhagic fever virus infection of rhesus macaques as a model of viral hemorrhagic fever: clinical characterization and risk factors for severe disease.

Simian Hemorrhagic Fever Virus (SHFV) has caused sporadic outbreaks of hemorrhagic fevers in macaques at primate research facilities. SHFV is a BSL-2 pathogen that has not been linked to human disease; as such, investigation of SHFV pathogenesis in non-human primates (NHPs) could serve as a model for hemorrhagic fever viruses such as Ebola, Marburg, and Lassa viruses. Here we describe the pathogenesis of SHFV in rhesus macaques inoculated with doses ranging from 50 PFU to 500,000 PFU. Disease severity was independent of dose with an overall mortality rate of 64% with signs of hemorrhagic fever and multiple organ system involvement. Analyses comparing survivors and non-survivors were performed to identify factors associated with survival revealing differences in the kinetics of viremia, immunosuppression, and regulation of hemostasis. Notable similarities between the pathogenesis of SHFV in NHPs and hemorrhagic fever viruses in humans suggest that SHFV may serve as a suitable model of BSL-4 pathogens.

Chimeric arteriviruses generated by swapping of the M protein ectodomain rule out a role of this domain in viral targeting.

Arteriviruses are enveloped, positive-strand RNA viruses for which the two major envelope proteins GP(5) and M occur as disulfide-linked heterodimers. These were assumed to serve the viral targeting functions, but recent ectodomain swapping studies with equine arteritis virus (EAV) indicate that the GP(5) protein does not determine arteriviral tropism. Here, we focused on the short, 13- to 18-residue ectodomain of the M protein. Using an infectious cDNA clone of the Lelystad virus isolate of porcine reproductive and respiratory syndrome virus (PRRSV), we substituted the genomic sequence encoding the M ectodomain by that of murine lactate dehydrogenase-elevating virus, EAV, and the US PRRSV-isolate, VR2332. Viable viruses with a chimeric M protein were obtained in all three cases, but for the latter two only after removal of the genomic overlap between the M and GP(5) genes. Characterization of the chimeric viruses revealed that they could be distinguished immunologically from wild-type virus, that they were genetically stable in vitro, but that they were impaired in their growth, reaching lower titers than the parental virus. The latter appeared to be due to an increased particle-to-infectivity ratio of the chimeric virus particles. Interestingly, the chimeric viruses had retained their ability to infect porcine cells and had not acquired tropism for cells susceptible to the viruses from which the foreign ectodomains were derived. We conclude that the surface structures composed by the arterivirus M and GP(5) ectodomains do not determine viral tropism.

Enzyme-linked immunosorbent assay for detection of antibodies against simian hemorrhagic fever virus.

Better assays are needed for the detection of simian hemorrhagic fever virus (SHFV), which induces persistent infection without overt signs of disease in most old world monkeys, but causes a fatal hemorrhagic fever in macaques. An enzyme-linked immunosorbent assay (ELISA) is described here that is useful in identifying primates previously exposed to SHFV. This assay involves testing serum samples against SHFV and cell antigens to obtain an ODvirus-to-ODcell ratio that eliminates potential high background values associated with primate serum. High correlation was found using this assay, compared with that found with the current "gold standard" indirect immunofluorescence assay (IFA). However, this ELISA is less time consuming, less subjective, and not as prone to human error than the SHFV-IFA.

Autoantibodies against golgi apparatus induced by arteriviruses.

Members of the genus Arterivirus within the monogeneric family Arteriviridae are lactate dehydrogenase-elvating virus (LDV), porcine reproductive and respiratory syndrome virus (PRRSV), equine arteritis virus (EAV) and simian hemorrhagic fever virus. In LDV-infected mice the appearance of autoantibodies against Golgi-antigen dominated the early immune response. Shared antigenicity between LDV and Golgi-antigen of normal cells could not be demonstrated. Monoclonal antibodies (MAbs) reacted either with LDV or with Golgi-antigen but not with both. Immunization of mice with the porcine arterivirus PRRSV, however, led to the establishment of MAbs that recognized the structural glycoprotein GP3 as well as Golgi-antigen of normal porcine cells indicating molecular mimicry of viral and cellular antigen. In addition to cross-reactive antibodies MAbs solely reactive with Golgi-antigen were observed. After immunization of mice with EAV, the equine arterivirus, clones were isolated producing Golgi-antigen recognizing autoantibodies. Morphogenesis of arteriviruses occurs in the Golgi region. The autoimmune responses following immunization with arteriviruses may offer an approach for determining the mechanism by which such responses develop and become of biologic importance.

Effect of dietary energy source and immunological challenge on growth performance and immunological variables in growing pigs.

Forty-eight growing pigs (23 kg BW) were assigned to four treatments (n = 12) arranged as a 2 x 2 factorial. Dietary energy source (conventional [CON] vs high-oil corn [HOC]), with or without an immunological challenge (IC) regimen constituted main effects. The IC regimen consisted of injection of endotoxin (E. coli lipopolysaccharide [LPS]) and vaccination for porcine respiratory and reproductive syndrome (PRRS). Growth performance data were collected over a 5-wk period and are presented as prechallenge (d 1 to 14; d 1 was the 1st d of the study), challenge (d 15 to 21), and postchallenge (d 22 to 36) periods, and overall. Overall, the pigs fed HOC consumed less feed (P < .11) and gained more efficiently (P < .03). During the immunological challenge period, ADG was depressed 21% and feed intake 15% (P < .01). The IC resulted in lower (P < .01) serum alpha-1-acid glycoprotein (AGP) concentrations on d 22, and the magnitude of the reduction was greater in the pigs fed the CON diet (energy source x immune challenge, P < .10). Serum AGP concentrations remained lower (P < .08) in challenged pigs on d 36. Immunoreactive prostaglandin concentrations were higher (55%, P < .08) in the pigs fed HOC immediately following the IC period (d 22). The data reported herein indicate that the performance of pigs fed HOC is satisfactory, and that feeding HOC does not compromise growth performance during or after an immunological challenge.

Proteins encoded by open reading frames 3 and 4 of the genome of Lelystad virus (Arteriviridae) are structural proteins of the virion.

Four structural proteins of Lelystad virus (Arteriviridae) were recognized by monoclonal antibodies in a Western immunoblotting experiment with purified virus. In addition to the 18-kDa integral membrane protein M and the 15-kDa nucleocapsid protein N, two new structural proteins with molecular masses of 45 to 50 kDa and 31 to 35 kDa, respectively, were detected. Monoclonal antibodies that recognized proteins of 45 to 50 kDa and 31 to 35 kDa immunoprecipitated similar proteins expressed from open reading frames (ORFs) 3 and 4 in baculovirus recombinants, respectively. Therefore, the 45- to 50-kDa protein is encoded by ORF3 and the 31- to 35-kDa protein is encoded by ORF4. Peptide-N-glycosidase F digestion of purified virus reduced the 45- to 50-kDa and 31- to 35-kDa proteins to core proteins of 29 and 16 kDa, respectively, which indicates N glycosylation of these proteins in the virion. Monoclonal antibodies specific for the 31- to 35-kDa protein neutralized Lelystad virus, which indicates that at least part of this protein is exposed at the virion surface. We propose that the 45- to 50-kDa and 31- to 35-kDa structural proteins of Lelystad virus be named GP3 and GP4, to reflect their glycosylation and the ORFs from which they are expressed. Antibodies specific for GP3 and GP4 were detected by a Western immunoblotting assay in swine serum after an infection with Lelystad virus.

Detection of porcine reproductive and respiratory syndrome virus and Mycoplasma hyorhinis antigens in pulmonary lesions of pigs suffering from respiratory distress.

Eleven field cases of a disease characterized by severe dyspnoea or abdominal breathing were examined post mortem. The affected pigs had antibody against porcine reproductive and respiratory syndrome virus (PRRSV). The predominant lung lesions were severe proliferative and interstitial pneumonia, and slight suppurative bronchopneumonia. The lesions were closely associated with the sites at which PRRSV and Mycoplasma hyorhinis antigens were detected. Four of five pigs inoculated with PRRSV developed slight pneumonitis. The fifth animal, which died of severe pneumonitis, yielded a heavy culture of M. hyorhinis. These findings demonstrate that dual infection with M. hyorhinis and PRRSV caused severe pulmonary lesions.

An ELISA for porcine reproductive and respiratory syndrome: production of antigen of high quality.

A reliable method was developed to produce a viral antigen preparation from porcine reproductive and respiratory syndrome virus (PRRSV) infected MARC-145 cells by solubilizing the virus with Triton X-100. This method eliminated problems previously encountered with high background reactions associated with PRRSV antigen or cell control antigen. With this new antigen, an indirect enzyme-linked immunosorbent assay (ELISA) was adapted to detect swine serum anti-body against PRRSV. In the ELISA, non-specific reactions associated with test serum samples have been eliminated by utilizing an effective blocking serum diluent. The ELISA is more sensitive than an indirect immunofluorescent assay (IFA), particularly with late-infection sera, while maintaining a high diagnostic specificity. In a comparison of IFA and ELISA using sera collected from 250 pigs of various ages from 5 herds that had PRRS histories, IFA revealed 178 positive samples and 72 negative samples. All of the IFA-positive sera were proven to be ELISA reactors. However, nearly one-half (34/72) of the IFA-negative samples were also ELISA reactors. The diagnostic specificity and sensitivity of the ELISA were 100% and 96.6% with 257 serum samples collected from known healthy PRRS-negative swine herds and 57 sera collected from infected swine at 6 to 56 days after infection, respectively. The ELISA is technically superior to IFA, time-efficient and cost-effective, and suitable for testing of a large number of samples over a short period of time.

Detection of antibodies against porcine reproductive and respiratory syndrome (PRRS) virus in swine sera by enzyme-linked immunosorbent assay.

We developed an enzyme-linked immunosorbent assay (ELISA) to detect antibodies against PRRS virus in swine sera. The ELISA antigen was prepared from MARC-145 cells infected with PRRS virus. The results from serial serum samples from experimentally infected and random swine indicated that the ELISA was more sensitive than indirect fluorescent and immunoperoxidase monolayer assays. Since the ELISA enables many sera to be simultaneously and rapidly tested, it was useful for detection antibodies against PRRS virus in swine sera.

Comparison of a commercial ELISA and an immunoperoxidase monolayer assay to detect antibodies directed against porcine respiratory and reproductive syndrome virus.

A commercially available enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against porcine respiratory and reproductive syndrome virus (PRRSV) was compared to an immunoperoxidase monolayer assay (IPMA). Serum samples used were collected from pigs experimentally infected with either the American or European antigenic type of PRRSV, and also from piglets born to sows that had been experimentally infected with the European antigenic type of PRRSV. In addition, three sets of European field sera (n = 275, n = 68, n = 349) were tested and evaluated using the IPMA as the gold standard. Results showed that both the IPMA and the ELISA were able to detect antibodies against the two antigenic types of PRRSV. When sera of experimentally infected pigs were tested, the IPMA with homologous antigen detected antibodies 2 to 3 days earlier than the ELISA, and was more sensitive in detecting maternal antibodies. The ELISA was slightly more sensitive for detecting antibodies against the American type than for the European type. When sets of field sera were tested, the relative sensitivity of the ELISA ranged between 0.68 and 0.91, and the relative specificity ranged between 0.75 and 0.97. However, in two of these sets (n = 275, n = 349) we determined that a decrease of the threshold value of ELISA (from 0.4 to 0.3) increased sensitivity without loss of specificity. We concluded that the ELISA is an easy, quick and reliable test to diagnose PRRSV infection in swine herds.

Comparison of the antigen distribution of two US porcine reproductive and respiratory syndrome virus isolates with that of the Lelystad virus.

One hundred 4-week-old cesarean-derived colostrum-deprived pigs were inoculated with one of two different US porcine reproductive and respiratory syndrome virus (PRRSV) isolates (VR2385, VR2431) or the European Lelystad virus to detect and compare the location and amount of virus antigen. Interstitial pneumonia, myocarditis, lymphadenopathy, and encephalitis were consistently seen in all three groups; however, disease and lesions were more severe in the VR2385 group. Immunohistochemical evaluation of formalin-fixed tissues revealed virus antigen in alveolar macrophages in lungs of 22/25, 14/25, 14/25, and 0/25 of the VR2385, VR2431, Lelystad, and control pigs, respectively. Follicular macrophages and dendritic cells in the lymph nodes of 14/25, 10/25, 10/25, and 0/25 pigs from the VR2385, VR2431, Lelystad, and control groups, respectively, stained positive for virus antigen. Similar cells in the tonsils from 25/25, 21/25, 23/25, and 0/25 pigs from the VR2385, VR2431, Lelystad, and control groups, respectively, stained positive for virus antigen. Other tissues and cells in which virus antigen was detected included macrophages and endothelial cells in the heart, macrophages, and interdigitating cells in the thymus, macrophages and dendritic cells in the spleen and Peyer's patches, and macrophages in hepatic sinusoids, renal medullary interstitium, and adrenal gland. PRRSV persisted in macrophages in the lung, tonsil, lymph node, and spleen for at least 28 days. Significantly more PRRSV antigen was detected in the lung (P < 0.01), lymph nodes (P < or = 0.05), and tonsils (P < 0.05) of the VR2385 pigs than was detected in the same tissues of the VR2431 and Lelystad pigs. The cell types in which PRRSV antigen was detected and the distribution of PRRSV antigen-positive cells within particular tissues and organs were generally similar for the different virus inoculation groups despite differences in virulence of the isolates.

Mycoplasma hyorhinis infection levels in lungs of piglets with porcine reproductive and respiratory syndrome (PRRS).

The infection levels of Mycoplasma hyorhinis, M. hyopneumoniae and M. hyosynoviae in the lung of piglets were examined in relation to porcine reproductive and respiratory syndrome (PRRS). These animals consisted of 43 PRRS piglets with PRRS, 2 piglets infected with PRRS virus but symptom-free, and 10 control piglets free of PRRS virus and its antibody. M. hyorhinis was isolated from 40 of the 43 PRRS piglets, from 1 of the 2 latent infected piglets and from 3 of the 10 control piglets. The number of M. hyorhinis isolated from the lungs of PRRS piglets was more than 10(5) CFU/g, but those isolated from the latent infected piglets and the control piglets were less than 10(3) CFU/g. In addition to this, Haemophilus parasuis and Pasteurella spp. were frequently isolated from the piglets with PRRS (51.2% and 25.6%, respectively). On the other hand, M. hyopneumoniae was isolated from only 4 of 55 piglets tested, and M. hyosynoviae was not isolated. M. hyorhinis was also detected directly in the lung emulsion samples from almost all the PRRS piglets using a polymerase chain reaction-based method.

Improved performance of a large pig complex after sequential nursery depopulation.

An attempt was made to compare the productivity and financial benefits of nursery depopulation on five large pig farms which were all part of one complex. Each farm had been experiencing poor post weaning performance for 12 months, and had previously been infected with porcine reproductive and respiratory syndrome virus (PRRSV). A plan to depopulate each nursery sequentially was established, and the pigs were moved to fattening facilities on one of the farms (farm 3) where space was available. Over a four week period, the nurseries of farms 1, 2, 4 and 5 were emptied, cleaned and disinfected, and any changes in nursery performance, mortality and the seroprevalence of antibodies to PRRSV were then assessed for one year. The financial benefit to the entire farm complex was analysed by using partial budget methods. During the year a net benefit of $1,708,431 was assessed to the farm complex owing to the increased numbers of marketable pigs and the reduced antibiotic costs. There were highly significant improvements in nursery growth rate and decreases in mortality on farms 1, 2, 4 and 5, and antibodies to PRRSV were detected on farms 3 and 4 but not on farms 1, 2 and 5. The inability to empty the farm 3 fattening facility, which housed the pigs from the other sites, may have led to the maintenance of its PRRSV positive status and could have served as the source of virus for farm 4.