Adenovirus-mediated expression of interferon-alpha delays viral replication and reduces disease signs in swine challenged with porcine reproductive and respiratory syndrome virus.

In this study, pigs were injected with a nonreplicating human adenovirus type 5 vector expressing porcine interferon-alpha (Ad5-pIFN-alpha) and then challenged with porcine reproductive and respiratory syndrome virus (PRRSV) to determine whether the presence of increased levels of IFN-alpha would decrease viral replication and/or disease. Groups of 10 pigs each were inoculated with Ad5-pIFN-alpha and not challenged, Ad5-pIFN-alpha and challenged with PRRSV 1 d later, or inoculated with a control adenovirus that does not express IFN-alpha (Ad5-null) and challenged 1 d later with PRRSV. IFN-alpha levels in all pigs inoculated with the Ad5-pIFN-alpha were elevated the day of challenge (1 d after inoculation), but were undetectable by 3 d after inoculation in the pigs that were not challenged with PRRSV. Pigs inoculated with Ad5-pIFN-alpha and challenged with PRRSV had lower febrile responses, a decreased percentage of lung involvement at 10 d post-infection, delayed viremia and antibody response, and higher serum IFN-alpha levels as a result of PRRSV infection, compared to pigs inoculated with Ad5-null and challenged with PRRSV. These results indicate that IFN-alpha can have protective effects if present during the time of infection with PRRSV.

Genomic characterization of virulent, attenuated, and revertant passages of a North American porcine reproductive and respiratory syndrome virus strain.

Pigs were exposed to three passages of the NADC-8 strain of porcine reproductive and respiratory syndrome virus (PRRSV) to investigate the relationship between genotypic and phenotypic properties. Differences were found in the virulence of the three passages called virulent, intermediate, and avirulent. Avirulent virus was derived by attenuation of virulent virus in cell culture and intermediate virus was derived by passage of avirulent virus in a pig. Nucleotide sequence differences between virulent and avirulent virus consisted of 50 nucleotide changes and a three-nucleotide deletion, and between avirulent and intermediate virus consisted of 8 nucleotide changes resulting in six amino acid changes. Three of these amino acid changes were direct reversions to virulent virus. Genetic changes, especially those seemingly associated with attenuation followed by some degree of reversion to virulence, in ORF1a, ORF1b, and ORF 6 regions of the genome may be involved in the control of PRRSV replication and virulence.

In vitro detection of Shiga toxin using porcine alveolar macrophages.

Porcine alveolar macrophages were found to be highly susceptible to the cytolytic effects of a toxin (Shiga toxin [Stx]) produced by certain strains of Escherichia coli and sometimes associated with clinical disease in pigs and other animals. In comparison with the cells that are most commonly used for Stx detection and titration in vitro (namely, Vero cells), porcine alveolar macrophages appeared to be generally more sensitive and test results could be obtained in less time. Moreover, unlike Vero cells, porcine alveolar macrophages need not be continuously propagated to ensure immediate availability. They can simply be removed from a low-temperature repository, thawed, seeded, and shortly thereafter exposed to the sample in question. These characteristics suggest that porcine alveolar macrophages may be useful in developing a highly sensitive and timely diagnostic test for Stx.

The effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance.

From a worldwide perspective, porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) are the most common viral causes of porcine reproductive failure. A typical epidemic of PPV-induced reproductive failure is presented as an increased number of mummified fetuses and sometimes, entire litters are mummified. If infection with PPV is very early in gestation, the number of liveborn pigs may be further reduced as a result of embryonic death and resorption. During the acute stage of infection gilts and sows have few, if any, clinical signs, and it is unlikely that PPV is ever the direct cause of abortion. In contrast, a typical epidemic of PRRSV-induced reproductive failure is presented as a broader spectrum of clinical features including abortions, late-term dead fetuses, stillborn pigs, and weakborn pigs. In the later stages of an epidemic, there may also be an increase in the number of mummified fetuses, but their prevalence is likely to be far less than during an epidemic of PPV-induced reproductive failure. During the acute stage of infection with PRRSV, gilts and sows may have few, if any, clinical signs, or they may be severely affected and even die. This difference largely reflects the relative virulence of the strain of PRRSV causing the epidemic. A timely and reliable laboratory diagnosis of either disease can be made when appropriate tests are performed with appropriate samples. Vaccines are available for prevention of both diseases.

Safety and efficacy of vaccination of pregnant gilts against porcine reproductive and respiratory syndrome.

OBJECTIVE: To determine the safety and efficacy of vaccination of pregnant gilts with an attenuated strain of porcine reproductive and respiratory syndrome virus (PRRSV). ANIMALS: 16 pregnant gilts. PROCEDURE: Pregnant gilts free of antibodies for PRRSV were assigned (4 gilts/group) to the following groups: group I, untreated controls; group II, vaccinated on day 60 of gestation; group III, vaccinated on day 60 of gestation and exposed to virulent PRRSV on day 90 of gestation; and group IV, exposed to virulent PRRSV on day 90 of gestation. Safety and efficacy of vaccination was evaluated by group comparisons of prenatal and postnatal survival of fetuses and pigs, respectively, and by the condition and rate of weight gain of liveborn pigs. RESULTS: Collective (prenatal and postnatal) death losses up to day 15 after farrowing (conclusion of study) were similar for groups I (7/47, 14.9%) and II (7/44, 16.9%) but were greater for group III (18/49, 36.7%) and were greater still for group IV (23/37, 62.2%). Mean body weight 15 days after farrowing was greatest for pigs in litters of group I (4.46 kg) and progressively less for the other groups (3.87, 3.76, and 2.18 kg for groups II, III, and IV, respectively). CONCLUSIONS: Using these conditions, vaccination of gilts during midgestation appeared to be safe. However, it provided only partial protection against subsequent exposure to virulent virus. CLINICAL RELEVANCE: Attenuated-PRRSV vaccines may have to be administered to naive gilts > 30 days before conception to provide maximum protection throughout gestation.

Evidence for divergence of restriction fragment length polymorphism patterns following in vivo replication of porcine reproductive and respiratory syndrome virus.

OBJECTIVE: To determine stability of the restriction fragment length polymorphism (RFLP) pattern of a porcine reproductive and respiratory syndrome vaccine virus and patterns of other viral strains as they replicate in pigs. SAMPLE POPULATION: Field samples of porcine reproductive and respiratory syndrome virus (PRRSV) and samples from 2 weaned pigs, 2 nursery-age pigs, and 5 gilts experimentally infected with PRRSV. PROCEDURE: PRRSV was isolated from field samples, experimentally infected pigs, or pigs that were in contact with experimentally infected pigs. For each virus, RNA was isolated from infected cells, and RFLP patterns were determined. RESULTS: 61% of field samples had 2-5-2 RFLP patterns characteristic of the vaccine virus, 32% had field virus RFLP patterns, and 7% had intermediate RFLP patterns that indicated a virus with a close relationship to the vaccine virus. Viruses isolated from experimentally infected pigs had no change in RFLP patterns after up to 13 weeks of in vivo replication and transmission to contact pigs. CONCLUSIONS AND CLINICAL RELEVANCE: RFLP patterns distinguish the vaccine and field strains of PRRSV; however, as the vaccine virus spreads among a swine population, the RFLP pattern can change to a related intermediate pattern. A glycine at residue 151 of open reading frame 5 is another marker for the vaccine virus; this glycine is rapidly lost and eventually replaced with arginine as the vaccine virus replicates in pigs.

Identification and clinical assessment of suspected vaccine-related field strains of porcine reproductive and respiratory syndrome virus.

OBJECTIVE: To determine the origin and clinical relevance of selected strains of porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV). ANIMALS: 38 pigs without antibodies for PRRSV. PROCEDURE: A seemingly uncommon restriction endonuclease digestion site in a commercially available vaccine strain of attenuated PRRSV was tested for its stability and prevalence under defined conditions. Selected field strains of PRRSV, with or without the restriction-site marker, were subsequently tested in pigs for virulence and for their ability to replicate competitively in pigs simultaneously given the vaccine. RESULTS: Under experimental conditions, the restriction-site marker was stable during long-term infection of pigs. It was not detected in any of the 25 field strains of PRRSV that were isolated before use of the vaccine or 21 of 25 field strains that were isolated after use of the vaccine but that, on the basis of previous testing, were believed unrelated to the vaccine strain. Conversely, it was detected in 24 of 25 field strains that were isolated after use of the vaccine and that, on the basis of previous testing, were believed to be direct-line descendants of the vaccine strain. Putative vaccine-related strains caused more pronounced pathologic changes than did the vaccine strain alone, and they predominated during replication in pigs also given the vaccine strain. CONCLUSIONS: In some swine herds, the vaccine strain may have persisted and mutated to a less attenuated form. CLINICAL RELEVANCE: The potential for persistence and mutation of specific strains of virus should be an important consideration when designing vaccination programs involving attenuated PRRSV.

Diagnostic implications of concurrent inoculation with attenuated and virulent strains of porcine reproductive and respiratory syndrome virus.

OBJECTIVE: To determine the predominant strain of progeny virus in samples obtained from cell cultures and pigs exposed simultaneously to attenuated and virulent strains of porcine reproductive and respiratory syndrome virus (PRRSV). SAMPLE POPULATION: Cell cultures and twenty 4-week-old pigs. PROCEDURE: Cell cultures and pigs were simultaneously exposed to various relative concentrations of an attenuated, cell-culture-adapted vaccine strain and a virulent field strain of PRRSV. Progeny virus obtained at selected intervals thereafter was tested to determine strain identity by use of restriction fragment length polymorphism (RFLP) analysis. RESULTS: Progeny virus from infected cell cultures comprised the attenuated strain, alone or in combination with the virulent strain, except when cultures had been exposed to a large excess (> 100,000-fold) of the virulent strain. Progeny virus from infected pigs comprised only the virulent strain regardless of the relative concentrations of the 2 strains to which the pigs had been exposed. CONCLUSIONS: During concurrent replication in cell cultures, the attenuated strain quickly predominated. Conversely, during concurrent replication in pigs, the virulent strain quickly predominated. CLINICAL RELEVANCE: It is unlikely that only an attenuated strain of PRRSV would be identified by RFLP testing of samples obtained from pigs concurrently infected with a virulent strain of PRRSV. Nevertheless, the ability of a cell-culture-adapted attenuated strain of PRRSV to predominate during cell culture passage (the first step in the current RFLP testing procedure) indicated that, if possible, samples should be obtained from pigs that do not have a history of direct or indirect exposure to attenuated-virus vaccine.

Clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (PRRS) virus isolated from field cases of "atypical" PRRS.

OBJECTIVE: To determine clinical consequences of exposing pregnant gilts to strains of porcine reproductive and respiratory syndrome (PRRS) virus (PRRSV) isolated from field cases of "atypical" or "acute" PRRS in vaccinated herds. ANIMALS: 20 pregnant gilts and their pigs and fetuses. PROCEDURE: 8 pregnant gilts (principals: 4 groups [2 gilts/group]) were exposed oronasally at or about 45 days of gestation to 1 of 4 strains of PRRSV and necropsied 6 weeks later. Nonexposed controls (2 additional pregnant gilts) were kept under otherwise similar conditions. The experiment was repeated, except that principals were exposed at or approximately 90 days of gestation and allowed to farrow. Clinical observations were made at least twice daily, and samples and specimens from gilts and their fetuses and pigs were tested for PRRSV and homologous antibody. RESULTS: Exposure of pregnant gilts to PRRSV at or approximately 45 days' gestation resulted in low prevalence of transplacental infection and fetal death. Exposure of pregnant gilts to PRRSV at or approximately 90 days' gestation resulted in higher prevalence of transplacental infection and fetal death. Moreover, 1 gilt aborted and many liveborn pigs of other litters were weak and unthrifty. Clinical signs of disease and reproductive failure were especially severe for a field strain of PRRSV isolated from an epizootic that fit the strictest definition of atypical PRRS. Controls remained clinically normal and free of PRRSV. CONCLUSION AND CLINICAL RELEVANCE: Some strains of PRRSV now circulating in US swine herds are more virulent than those encountered in the past. Clinical PRRS in vaccinated herds suggests need for a new generation of vaccines.

Clinical effects of porcine reproductive and respiratory syndrome virus on pigs during the early postnatal interval.

OBJECTIVE: To determine the effect of congenital and early postnatal infection of pigs with porcine reproductive and respiratory syndrome virus (PRRSV) on postnatal survival and growth. ANIMALS: 20 pregnant gilts and their pigs and fetuses. PROCEDURE: 16 pregnant gilts (principals) comprising 4 groups (4 gilts/group) were exposed oronasally to 4 strains of PRRSV (a vaccine strain, and 3 field strains) at or about day 90 of gestation. Four pregnant gilts (controls) were kept under similar conditions, except for exposure to PRRSV. Samples collected from pigs before ingestion of colostrum and samples and specimens collected from pigs at selected times thereafter were tested for PRRSV and homologous antibody. Pigs were observed for clinical signs and were weighed at birth and at weekly intervals until they were euthanatized and necropsied at about 3 weeks of age. RESULTS: At least some members of all litters of principal gilts were infected congenitally. Most noninfected, liveborn littermates became infected within the first week of life. Infection of pigs with field strains did, and infection of pigs with the vaccine strain did not, adversely affect postnatal survival and growth rate. All infected pigs had generalized lymph node enlargement. CONCLUSION: Exposure of pregnant gilts to either attenuated (vaccine) or virulent (field) strains of PRRSV can result in congenital infection. Vaccine as well as field strains can be transmitted postnatally from infected to noninfected littermates. Pigs infected with field strains have a poorer rate of survival and growth than do noninfected pigs. CLINICAL RELEVANCE: Because attenuated (vaccine) PRRSV can cause congenital infection and be transmitted postnatally from congenitally infected to immune-naive pigs, the use of attenuated virus during gestation is, at best, questionable.

The role of biotechnologically engineered vaccines and diagnostics in pseudorabies (Aujeszky's disease) eradication strategies.

Modern-day biotechnology has an almost unlimited number of possibilities for reducing the impact of hereditary and infectious diseases. To date one of its most visible and rewarding applications for veterinary medicine has been in the genetic engineering of vaccines and diagnostics to assist in the eventual eradication of pseudorabies (PR, Aujeszky's disease). In the following review we summarize some of the most pertinent issues relative to PR eradication and point out the present and potential role of biotechnology in achieving our goal.

Genetic variation and phylogenetic relationships of 22 porcine reproductive and respiratory syndrome virus (PRRSV) field strains based on sequence analysis of open reading frame 5.

Porcine reproductive and respiratory syndrome virus (PRRSV) strains from 13 states in the United States, Guatemala and Canada were used to compare the envelope glycoprotein gene (ORF 5) nucleotide and deduced amino acid sequences. The gene was the same size, 603 nt, for all the 22 field strains. These strains had 89-94% amino acid identity compared to reference strain VR 2332. A putative signal sequence and cleavage site between residues 31 and 32 was identified and three potential glycosylation sites were present on all but two strains. Hydrophobicity/hydrophilicity and surface probability analyses reveal a primary structure for the envelope glycoprotein (E protein) with six potential surface regions that could be antigenic sites. Similar E protein structural features are conserved for the prototype European PRRSV-Lelystad virus.

Comparison among strains of porcine reproductive and respiratory syndrome virus for their ability to cause reproductive failure.

OBJECTIVE: To compare the virulence of selected strains of porcine reproductive and respiratory syndrome virus (PRRSV) relative to reproductive performance of pregnant gilts. DESIGN: 16 pregnant gilts (principals) were exposed oronasally to 4 strains (vaccine strain RespPRRS, field strains VR-2385, VR-2431, and NADC-8, 4 gilts/strain) of PRRSV on or about day 90 of gestation, 4 pregnant gilts (controls) were kept under similar conditions, except for exposure to PRRSV. Samples and specimens obtained from gilts, pigs (before ingestion of colostrum), and fetuses were tested for PRRSV and homologous antibody. ANIMALS: 20 pregnant gilts. PROCEDURE: The virulence of each strain of PRRSV was evaluated mainly on the clinical status of the corresponding litters at farrowing. RESULTS: Most gilts remained clinically normal throughout the study and farrowed normally at or near the expected farrowing time. All virus strains crossed the placenta of principal gilts to infect fetuses in utero. The number of late-term dead fetuses (which appeared to be the best measure of relative virulence) ranged from 0 for litters of control gilts and gilts exposed to strain RespPRRS, to 38 for gilts exposed to strain NADC-8. All principal gilts became viremic and developed antibody against PRRSV. All strains persisted in alveolar macrophages of at least some principal gilts for at least 7 weeks after exposure. CONCLUSION: Strains of PRRSV vary in virulence. CLINICAL RELEVANCE: The effects of PRRSV on reproductive performance are strain dependent and this should be considered in making a tentative diagnosis on the basis of clinical observations.

Diagnosis of porcine reproductive and respiratory syndrome using infected alveolar macrophages collected from live pigs.

A highly sensitive method of detecting infection of live pigs with porcine reproductive and respiratory syndrome virus (PRRSV) was developed by testing alveolar macrophages collected by pulmonary lavage. Five pigs were exposed by oronasal inoculation or by contact to PRRSV when they were 10 (1 pig) or 14 weeks (4 pigs) of age. Diagnostic samples (alveolar macrophages and sera) were collected from each pig just before exposure to PRRSV. During the next 9 weeks sera were collected at weekly intervals and alveolar macrophages were collected at weeks 2 and 4-9. Both sera and alveolar macrophages were suitable for detecting early infection, but alveolar macrophages were clearly the better sample after longer intervals. Virus was last isolated from serum at week 4 (from 1 of 5 pigs), whereas it was isolated from the alveolar macrophages of 4 of the 5 pigs at week 4 and from at least 2 pigs at each of the weekly intervals thereafter (i.e. weeks 5, 6, 7, 8, and 9 postexposure). The most sensitive method of testing alveolar macrophages for PRRSV was cocultivation with MARC-145 cells. None of the pigs had any clinical signs after exposure to PRRSV or as a result of pulmonary lavage and there was no evidence that repeated pulmonary lavage caused anything other than a mild, transient (mild hyperemia) tissue reaction.

Diagnosis of porcine reproductive and respiratory syndrome.

The most suitable tissue samples and test procedures for the etiologic diagnosis of porcine reproductive and respiratory syndrome (PRRS) were found to depend on several variables including the age of the pig from which tissues were collected, the stage of infection (acute or persistent), the available complement of diagnostic reagents, and the urgency in obtaining results. When the diagnosis involved acute infection of congenitally or neonatally infected pigs, and susceptible cell culture(s) was available for virus isolation, then both serum and alveolar macrophages (AM) were reliable samples. Alveolar macrophages flushed from infected lungs provided a temporal advantage, however, in that in addition to their use for virus isolation, i.e., from a lysate of AM, they could be cultured in vitro and examined for the presence of viral antigens by immunofluorescence microscopy (FA) as early as 1 hour after they were added to the culture vessel. The examination of AM in this manner also circumvented the need for additional cell cultures to test for infectious virus. Testing presuckling sera by indirect FA for antibodies to PRRS virus also was of diagnostic value and, like FA with AM, could be completed soon after sample collection. For older pigs, AM were more reliable than serum, lungs, or any of 27 other tissues evaluated as diagnostic samples and were often the only samples in which infectious virus and viral antigens were detected when pigs were euthanized more than 3 weeks postexposure. A simple procedure for on-farm collection of AM as well as methods for testing AM for viral antigens and neonatal (presuckling) sera for homologous antibody in a modestly equipped laboratory, such as one that might be maintained by a veterinary practitioner, are described and discussed.

Temporal characterization of transplacental infection of porcine fetuses with porcine reproductive and respiratory syndrome virus.

Pregnant gilts were exposed to porcine reproductive and respiratory syndrome virus (PRRSV) by IV inoculation at or about gestation day 30 (3 gilts), 50 (3 gilts), 70 (3 gilts), or 90 (5 gilts) to investigate the likelihood of transplacental infection with PRRSV at various stages of gestation. At or about 3, 6, and 9 weeks after exposure, gilts were either euthanatized while still pregnant or allowed to farrow. Gilts and pigs were observed for clinical signs of infection, and gilts, pigs, and fetuses were tested for PRRSV and homologous antibody. All gilts were healthy throughout the study, except that farrowing was sometimes difficult and prolonged, and 2 gilts failed to farrow the entire litter. One gilt farrowed on day 111 of gestation; all others farrowed on day 114 or later. Porcine reproductive and respiratory virus was isolated from significantly (chi 2 test, P < 0.01) more fetuses and live and stillborn pigs of the 5 gilts that were infected at 90 or 92 days of gestation than from the fetuses and live and stillborn pigs of the 9 gilts that were infected at 72 or fewer days of gestation (ie, 33 of 44, 75% vs 3 of 78, 4%). After initial infection, PRRSV was isolated from gilts and their pigs for a maximum of 3 weeks and 8 to 11 weeks, respectively. Findings of this study, with regard to the temporal aspects of transplacental infection, may help explain why natural epizootics of PRRSV-induced maternal reproductive failure are often recognized principally as problems of late-term gestation and neonatal survival.

Size and antigenic comparisons among the structural proteins of selected autonomous parvoviruses.

The size and antigenic relationships among structural proteins (VPs) of canine parvovirus (CPV), feline parvovirus (FPV), porcine parvovirus (PPV), minute virus of mice (MVM) and bovine parvovirus (BPV) were determined by SDS-PAGE of radiolabelled, purified virus and immunoprecipitated viral proteins. Mature virions of CPV, FPV, PPV and MVM were composed of three VPs designated VP1, VP2 and VP3. The corresponding proteins of each virus were similar in molecular weight [79,000 to 82,500 (VP1), 65,000 to 66,000 (VP2), 62,000 to 63,500 (VP3)]. Additional similarities among VPs were indicated by antigenic relationships which included precipitation of VPs of CPV, FPV and PPV by both homologous antisera and antisera raised to each of the other two viruses and by precipitation of VPs of MVM by cat anti-FPV sera. A non-structural protein identified in lysates of cells infected with FPV and CPV was precipitated by cat anti-FPV and dog anti-CPV sera only. Mature virions of BPV were composed of four VPs [74,500 (VP1), 67,000 (VP2), 60,000 (VP3), 57,500 (VP4)] which were antigenically unrelated to those of the other parvoviruses tested. However, the possibility that swine are sometimes infected with a virus which is antigenically related to BPV was suggested by the finding that sera from conventionally raised swine, irrespective of their serological status for PPV, precipitated VPs of BPV, whereas neither pre-exposure sera nor anti-PPV sera from gnotobiotic pigs did so.