[Analysis of genome determinants of virulence and setting-up of a library of full-size genome copies of the attenuated virus strain of the porcine reproductive and respiratory syndrome virus North American type]. / Analiz genomnykh determinant virulentnosti i sozdanie biblioteki polnorazmernykh infektsionnykh kopii genoma attenuirovannogo shtamma virusa reproduktivno-repriratornogo sindroma svinei severoamerikanskogo tipa.

Primary genome structures of 3 variants of the NADC-8 North American virulent strain of porcine reproductive and respiratory syndrome virus (PRRSV) were compared for the purpose of detecting any potential genetic virulence determinants of genus Arterivirus. Apart from the virulent variant, we also investigated the attenuated variant, obtained after 251 passages in cell culture, and the intermediate variant isolated from a pig after a partial reversion of the attenuated virus. The attenuated variant genome acquired a 3-nucleotide deletion and 50 mutations versus its virulent precursor. A comparison of the attenuated and intermediary virus variants denoted 8 nucleotide mutations entailing substitutions of 6 amino acids in 3 open reading frames (ORF1a, ORF1b and ORF6). A 32-clone library was constructed in the pACYC177 plasmid vector, which comprised full-size copies of the genome of the NADC-8 attenuated variant strain (251), virus PPCC, for the purpose of experimentally verifying the functional role of the obtained mutations. Full-size analogues ((+)-chain of RNA) of the viral genome, comprising the CAP-structures and polyadenylated ones were obtained in vitro on the basis of the cloned DNA. Seven of the 8 analyzed clones of the viral genome were infected and their insertion into the MARC-145 cell resulted in obtaining of infectious PRRSVs. Four of the constructed recombinant viruses had delayed growth parameters, and 3 of them were similar to the parental strain. The described technology (inverse genetics) would make it possible to introduce changes into the viral genome in applied and fundamental research of Arteriviruses.

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.

Investigation of porcine parvovirus among persons with hemophilia receiving Hyate:C porcine factor VIII concentrate.

BACKGROUND: Porcine clotting factor has been used for more than 15 years to treat severe bleeding episodes in persons with hemophilia who have antibodies to human clotting factor. In 1996, QC procedures revealed for the first time the presence of porcine parvovirus (PPV) in the product. This report describes an investigation to determine the extent of product contamination and to evaluate past recipients of porcine clotting factor (Hyate:C, Speywood Biopharm) for evidence of PPV infection. STUDY DESIGN AND METHODS: Stored specimens from 22 lots of previously released Hyate:C were tested for the presence of PPV DNA by PCR and nested PCR assays. Serum specimens from 98 recipients of Hyate:C and 24 controls who did not receive Hyate:C were tested for PPV antibodies by an immunofluorescence assay. RESULTS: PPV DNA was detected in product from 21 of the 22 lots of Hyate:C, primarily by nested PCR testing. In contrast, none of the serum specimens from the 98 Hyate:C recipients tested positive for PPV IgG antibodies. CONCLUSION: The risk of human disease from percutaneous exposure to low levels of PPV seems to be low. Nevertheless, the theoretical risk of human infection with PPV has led to manufacturing changes, including PCR screening of all porcine plasma, which are designed to eliminate this risk.

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.

A brief review of procedures and potential problems associated with the diagnosis of porcine reproductive and respiratory syndrome.

Experience has shown that, for a number of reasons, a diagnosis of porcine reproductive and respiratory syndrome (PRRS) is sometimes difficult. In this review we discuss: (1) field observations and laboratory tests that are useful in arriving at a definitive diagnosis; (2) the impact of so-called atypical PRRS on diagnostic procedures in North America; (3) the means by which diagnostic problems can often be circumvented by appropriate sample selection; and (4) methods used for presumptive identification of PRRS virus strains.

Evaluation of protective immunity in gilts inoculated with the NADC-8 isolate of porcine reproductive and respiratory syndrome virus (PRRSV) and challenge-exposed with an antigenically distinct PRRSV isolate.

OBJECTIVES: To determine whether intrauterine inoculation of porcine reproductive and respiratory syndrome virus (PRRSV) interferes with conception and whether exposure to one strain of PRRSV provides protection against challenge-exposure (CE) with homologous or heterologous strains of PRRSV. ANIMALS: 40 gilts. PROCEDURE: Gilts were inoculated by intrauterine administration of a PRRSV isolate (NADC-8) at breeding. Inoculated and noninoculated gilts were exposed oronasally to homologous (NADC-8) or heterologous (European isolate) PRRSV during late gestation. Specimens from gilts and fetuses were tested against CE virus. Lack of virus in gilts indicated protective immunity for the dam, in fetuses indicated protection of gilt from reproductive losses, and in both groups indicated complete protection. RESULTS: In the homologous CE group, interval from inoculation to CE ranged from 90 to 205 days, and protection was complete. In the heterologous CE group, interval from inoculation to CE ranged from 90 to 170 days, and protection was incomplete. The CE virus was detected in gilts necropsied 134 to 170 days after CE and in a litter necropsied 170 days after CE. CONCLUSIONS: Homologous protection can be induced in gilts by exposure to live PRRSV. Heterologous protection from reproductive losses can be induced in gilts by exposure to live PRRSV; however, this protection is incomplete and may have a shorter duration than homologous protection. CLINICAL RELEVANCE: Exposure of swine to enzootic PRRSV will provide protection against homologous PRRSV-induced reproductive losses. Extent and duration of protection against heterologous PRRSV may be variable and dependent on antigenic relatedness of the virus strains used for inoculation and CE.

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.

Restriction fragment length polymorphism analysis of strains of porcine reproductive and respiratory syndrome virus by use of a nested-set reverse transcriptase-polymerase chain reaction.

OBJECTIVE: To increase the timeliness and sensitivity of a procedure that uses viral nucleic acid amplification followed by restriction fragment length polymorphism (RFLP) analysis for identifying strains of porcine reproductive and respiratory syndrome virus (PRRSV). SAMPLE POPULATION: 24 strains of PRRSV. PROCEDURE: A nested-set reverse transcriptase-polymerase chain reaction (RT-PCR) was developed and compared with a nonnested-set RT-PCR for sensitivity in amplifying known quantities of infective PRRSV. Once reaction conditions were optimized, the nested-set RT-PCR was tested for effectiveness with 24 strains of PRRSV isolated from swine. RESULTS: The nested-set RT-PCR was 100- to 1,000-fold more sensitive than the nonnested-set RT-PCR, detecting as little as 1 infective unit of PRRSV/ml of sample. It also was generally as sensitive as the combination of steps, namely virus isolation or propagation and nonnested-set RT-PCR, currently used routinely for amplifying PRRSV prior to RFLP analysis, and it was effective for amplifying all of the 24 strains of PRRSV tested. Using this RT-PCR, all tests were completed within 1.5 days (including RFLP analysis), compared with the > 7 days often required for the currently used method involving virus isolation and propagation. CONCLUSIONS: The nested-set RT-PCR was generally as sensitive as the combination of methods now used for PRRSV amplification prior to RFLP analysis, and it can markedly reduce the time required for testing. CLINICAL RELEVANCE: Presumptive identification of PRRSV strains can be provided in a more timely manner by use of a nested-set RT-PCR.

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.

Prior infection of nursery-age pigs with porcine reproductive and respiratory syndrome virus does not affect the outcome of transmissible gastroenteritis virus challenge.

Thirty-six specific-pathogen-free pigs were weaned at 2 weeks of age and separated into 4 treatment groups (A-D, 9 pigs/group). Treatment groups B and D were infected with porcine reproductive and respiratory syndrome virus (PRRSV), whereas groups A and C remained uninfected. Two weeks later, 1 pig from each group was necropsied to assess gross lung involvement, and then the remaining group D PRRSV-infected pigs and the group C uninfected pigs were challenged at 4 weeks of age with transmissible gastroenteritis virus (TGEV) to determine if prior infection with PRRSV increased the severity of TGEV disease after challenge. One hundred percent morbidity but no mortality occurred in pigs following challenge. Clinically, pigs of both groups C and D were similar in terms of onset and severity of diarrhea. The serum antibody response to TGEV and the amount and duration of TGEV shedding after challenge was similar for both groups. Only a few pigs in each group had a transient fever postchallenge, and both group C and group D pigs began to recover and to gain weight at or near the end of the first week postchallenge. It was concluded that the clinical course of TGEV disease was not markedly affected by infection of pigs with TGEV 2 weeks after they had been infected with PRRSV.

Differentiation of a porcine reproductive and respiratory syndrome virus vaccine strain from North American field strains by restriction fragment length polymorphism analysis of ORF 5.

The suitability of restriction fragment length polymorphism (RFLP) analysis for differentiating a porcine reproductive and respiratory syndrome virus (PRRSV) vaccine strain from other North American field strains was investigated. Open reading frame 5 nucleotide sequence data of the vaccine virus, its parent strain VR-2332, and 22 other strains of PRRSV included in this study indicated that 3 restriction enzyme gel patterns characterize the vaccine virus and the parent strain genotype. The combined 3 RFLP patterns differentiate the vaccine and parent virus from other PRRSV strains. This test will be a valuable tool in epidemiologic studies and will be useful in identifying individual strains in cases of multistrain PRRSV infections.

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.

Successful pseudorabies vaccination in maternally immune piglets using recombinant vaccinia virus vaccines.

Three gilts were vaccinated with a NYVAC vaccinia recombinant expressing glycoprotein gD of pseudorabies virus (PRV) (NYVAC/gD). After farrowing, the piglets were allowed to nurse normally to obtain colostral immunity and then were divided into four groups, receiving NYVAC/gD, a NYVAC recombinant expressing glycoprotein gB of PRV (NYVAC/gB), an inactivated PRV vaccine (iPRV), or no vaccine. The piglets were vaccinated twice, three weeks apart beginning at approximately two weeks of age and later challenged with virulent PRV oronasally. Piglets that received NYVAC/gB or iPRV were the best protected based on lack of mortality, lower temperature responses, decreased weight loss and decreased viral shedding after challenge. These results indicate effective strategies for stimulating active immune response while still under the protection of maternal immunity.

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.

Vaccination with recombinant vaccinia virus vaccines expressing glycoprotein genes of pseudorabies virus in the presence of maternal immunity.

Piglets which had received colostral antibody to pseudorabie virus (PRV) were divided into four groups and inoculated with a NYVAC vaccinia recombinant expressing glycoprotein gD of PRV, a NYVAC recombinant expressing glycoprotein gB of PRV, an inactivated PRV vaccine, or no vaccine. The piglets were vaccinated twice, 3 weeks apart, beginning at approximately 2 weeks of age and later challenged with virulent PRV oronasally. All three vaccines protected similarly when no maternal antibody was present. Although all three vaccines induced some active immunity in piglets with maternal antibody, piglets receiving the NYVAC/gB vaccine were the only ones protected similarly whether or not they had maternal antibodies to PRV.