Michael J. McGinley

Porcine reproductive and respiratory syndrome virus: a persistent infection.

Persistent infection with porcine reproductive and respiratory syndrome virus (PRRSV) was shown in experimentally infected pigs by isolation of virus from oropharyngeal samples for up to 157 days after challenge. Four 4 week old, conventional, PRRSV antibody-negative pigs were intranasally inoculated with PRRSV (ATCC VR-2402). Serum samples were collected every 2 to 3 days until day 42 post inoculation (PI), then approximately every 14 days until day 213 PI. Fecal samples were collected at the time of serum collection through day 35 PI. Oropharyngeal samples were collected at the time of serum collection from 56 to 213 days PI by scraping the oropharyngeal area with a sterile spoon, especially targeting the palatine tonsil. Turbinate, tonsil, lung, parotid salivary gland, spleen, lymph nodes and serum were collected postmortem on day 220 PI. Virus isolation (VI) on porcine alveolar macrophage cultures was attempted on all serum, fecal and oropharyngeal samples, as well as tissues collected postmortem. Postmortem tonsil tissues and selected fecal samples were also assayed for the presence of PRRSV RNA by the polymerase chain reaction (PCR). Serum antibody titers were determined by IFA, ELISA and SVN. Virus was isolated from all serum samples collected on days 2 to 11 PI and intermittently for up to 23 days in two pigs. No PRRSV was isolated from fecal samples, but 3 of 24 samples were PCR positive, suggesting the presence of inactivated virus. Oropharyngeal samples from each pig were VI positive 1 or more times between 56 and 157 days PI. Oropharyngeal samples from 3 of 4 pigs were VI positive on days 56, 70 and 84 PI. Virus was isolated from one pig on day 157 PI, 134 days after the last isolation of virus from serum from this animal. Virus was isolated from oropharyngeal samples for several weeks after the maximum serum antibody response, as measured by IFA, ELISA and SVN tests. All tissues collected postmortem were VI negative and postmortem tonsil samples were also negative by PCR. An important element in the transmission of PRRSV is the duration of virus shedding. The results of this study provided direct evidence of persistent PRRSV infection and explain field observations of long-term herd infection and transmission via purchase of clinically normal, but PRRSV infected, animals. Effective prevention and control strategies will need to be developed in the context of these results.

Characterization of the humoral immune response to porcine reproductive and respiratory syndrome (PRRS) virus infection

The development of the humoral immune response against porcine reproductive and respiratory syndrome (PRRS) virus was monitored by an indirect fluorescent antibody (IFA) test, immunoperoxidase monolayer assay (IPMA), enzyme-linked immunosorbent assay (ELISA), and serum virus neutralization (SVN) test over a 105-day period in 8 pigs experimentally infected with ATCC strain VR-2402. Specific antibodies against PRRS virus were first detected by the IFA test, IPMA, ELISA, and the SVN test 9-11, 5-9, 9-13, and 9-28 days postinoculation (PI), respectively, and reached their maximum values by 4-5, 5-6, 4-6, and 10-11 weeks PI, respectively, thereafter. After reaching maximum value, all assays showed a decline in antibody levels. Assuming a constant rate of antibody decay, it was estimated by regression analysis that the ELISA, IFA, IPMA, and SVN antibody titers would approach the lower limits of detection by approximately days 137, 158, 324, and 356 PI, respectively. In this study, the immunoperoxidase monolayer assay appeared to offer slightly better performance relative to the IFA test, ELISA, and SVN test in terms of earlier detection and slower rate of decline in antibody titers. Western immunoblot analysis revealed that antibody specific for the 15-kD viral protein was present in all pigs by 7 days PI and persisted throughout the 105-day observation period. Initial detection of antibodies to the 19-, 23-, and 26-kD proteins varied among pigs, ranging from 9 to 35 days PI. Thereafter, the antibody responses to these 3 viral proteins of PRRS virus continued to be detected throughout the 105-day study period. These results clearly indicate that the 15-kD protein is the most immunogenic of the 4 viral proteins identified and may provide the antigenic basis for the development of improved diagnostic tests for the detection of PRRS virus antibodies.

Porcine reproductive and respiratory syndrome virus: routes of excretion.

Abstract This study was conducted to delineate potential sites of exit and duration of shedding of porcine reproductive and respiratory syndrome virus (PRRSV). Two experiments of 6 pigs each were conducted. Pigs were farrowed in isolation, weaned at 7 days of age, and housed in individual HEPA filtered isolation chambers. In each experiment, 3 pigs served as controls and 3 were inoculated intranasally with PRRSV (ATCC VR-2402) at 3 weeks of age. In a first experiment, on days 7, 14, 21, 28, 35, and 42 post inoculation (PI), pigs were anesthetized and intubated. The following samples were collected: serum, saliva, conjunctival swabs, urine by cystocentesis, and feces. Upon recovery from anesthesia, the endotracheal tube was removed, rinsed, and the rinse retained. In the second experiment, the sampling schedule was expanded and serum, saliva, and oropharyngeal samples were collected from day 55 to day 124 PI at 14 day intervals. Virus was isolated in porcine alveolar macrophages up to day 14 from urine, day 21 from serum, day 35 from endotracheal tube rinse, day 42 from saliva, and day 84 from oropharyngeal samples. No virus was recovered from conjunctival swabs, fecal samples, or negative control samples. This is the first report of isolation of PRRSV from saliva. Virus-contaminated saliva, especially when considered in the context of social dominance behavior among pigs, may play an important role in PRRSV transmission. These results support previous reports of persistent infection with PRRSV prolonged recovery of virus from tonsils of swine.

Studies of porcine reproductive and respiratory syndrome (PRRS) virus infection in avian species

Porcine reproductive and respiratory syndrome virus (PRRSV) is a recently recognized virus of swine. As a newly emerging virus, much of the basic information regarding PRRSV is in the process of discovery. We report three experiments with PRRSV in birds, and a fourth experiment to evaluate the infectivity and transmissibility of avian-derived PRRSV in swine. Experiment 1 compared the susceptibility of Muscovy ducks, Mallard ducks, guinea fowl, and chickens to PRRSV. Birds were exposed to PRRSV (ATCC VR-2402) in drinking water and virus isolation was attempted from feces collected from cages. Based on the duration of fecal shedding of the virus, this experiment showed that Mallard ducks were particularly susceptible to PRRSV. Experiment 2 was done in mallards to corroborate and augment the observations of experiment 1. Virus was isolated from pooled mallard feces up to 25 days post exposure (PE) and from the intestinal contents of 8 of 20 birds euthanized on day 38 PE. No gross or microscopic lesions were observed in ducks collected between 0 and 15 days PE. Experiment 3 evaluated the infectivity and transmissibility of mallard-derived PRRSV in mallards. A cage of mallards orally exposed to PRRSV shed the virus in feces. Exposure of a second cage of mallards to feces from the first cage resulted in fecal shedding of PRRSV by birds in cage two. In turn, exposure to feces from the second cage led to fecal shedding by mallards in a third cage. Experiment 4 assessed the infectivity and transmissibility of mallard-derived virus in swine. Pigs intranasally exposed to PRRSV isolaed from mallard feces in experiment 2 became viremic, seroconverted by ELISA, and transmitted the virus to sentinel swine. Collectively, these studies show that the possibility exists for avian species to be involved in the epidemiology of PRRSV. This is the first report of PRRSV infection in a species other than swine.

Failure to consider the antigenic diversity of porcine reproductive and respiratory syndrome (PRRS) virus isolates may lead to misdiagnosis.

Porcine reproductive and respiratory syndrome (PRRS) is a recently identified viral disease of swine that is a significant problem in swine producing regions throughout the world. Because of the potential economic impact of PRRS on swine production, extensive resources have been expended on the development of accurate diagnostic tests and efficacious vaccines. One of the potential obstacles to the success of these efforts is antigenic diversity among PRRS virus isolates. Antigenic diversity has been demonstrated between North American and European PRRS virus field isolates by using homologous and heterologous polyclonal swine antiserum in an immunoperoxidase monolayer assay. Antigenic differences between US and European PRRS virus isolates have also been demonstrated using a panel of 3 monoclonal antibodies (MAbs) specific for the 15kD protein of the PRRS virus: SDOW 17, which was generated against the PRRS virus isolate ATCC VR-2332, and EP147 and VO17, which were generated against the PRRS virus isolate SD1. All 3 MAbs reacted, as determined by fluorescence microscopy, with 63 of 63 US isolates originating from the north-central region of the United States. Additionally, MAb SDOW17 reacted with the Lelystad PRRS virus and 56 of 56 European isolates representing 8 different countries (Denmark, France, Germany, Italy, Luxemburg, The Netherlands, Spain, and The United Kingdom). However, neither MAb EP147 nor MAb VO17 reacted with any of these European isolates. Similar observations have been made by Canadian investigators, who reported that the same panel of MAbs reacted with 10 of 10 Canadian PRRS virus isolates. These observations suggest that MAb SDOW17 could be used as a universal diagnostic reagent for the detection of PRRS virus infection in clinical specimens. However, the following report indicates that North American isolates are antigenically more diverse than previously demonstrated and that it may not be possible to rely on a single MAb for diagnostic purposes.