Genome sequence and description of Blautia brookingsii SG772 sp. nov., a novel bacterial species isolated from human faeces.

An anaerobic isolate SG772 belonging to the genus Blautia was isolated from a healthy human faecal sample. When compared using 16s rRNA sequence identity, SG772 showed only 94.46% similarity with its neighbour species Blautia stercoris. As strain SG772 showed both phenotypic and genomic differences from other members of the type species within the genus Blautia, we propose the designation of SG772 as novel species 'Blautia brookingsii SG772T'.

Serological evidence for the co-circulation of two lineages of influenza D viruses in equine populations of the Midwest United States.

Influenza D virus (IDV) is a newly described influenza type of the Orthomyxoviridae virus family that was first isolated from diseased swine in 2011 and has subsequently been detected in cattle around the world in 2014. In addition, serological evidence for IDV infection in humans has been recently established. Despite all the progress, the full range of susceptible hosts for this novel virus has yet to be determined, but includes swine, bovine, small ruminants and human. This study was designed to determine if equine is a possible host to this newly emerging influenza virus. Three hundred and sixty-four equine serum samples were collected in 2015 from 141 farms within the Midwestern United States. Serum samples were examined using hemagglutination inhibition (HI) assay against two established IDV lineages (D/OK and D/660) and one IDV-related human ICV lineage (C/JHB). Results of this study showed 44 (44 of 364, 12%) samples positive for antibodies against D/OK, 39 (39 of 364, 11%) samples positive for antibodies against D/660, and 41 (41 of 364, 11%) samples positive for antibodies against C/JHB. A subset of these samples was further confirmed via microtitre neutralization (MN) assay. Our data demonstrated that horses are susceptible to two lineages of IDV, and that these viruses were present in equine populations throughout multiple Midwestern states of the United States. These findings continue to support the need for further surveillance of IDV viruses in agricultural species to work towards a better understanding of the full host range and natural reservoirs of influenza D virus.

Porcine epidemic diarrhea virus: An overview of current virological and serological diagnostic methods.

Porcine epidemic diarrhea virus (PEDV) is the causative agent of an acute, highly contagious, and severe enteric disease that leads to high mortality rates in suckling piglets. Therefore, accurate diagnosis of PEDV infection is critical for the implementation of control measures for the virus. Many diagnostic tests have been recently developed and are currently available for the detection of PEDV, its proteins or nucleic acid, including virus isolation, immunofluorescence (IF) or immunohistochemistry (IHC), polymerase chain reaction (PCR) and isothermal amplification assays. Additionally, several serological assays have been developed and are currently used for the detection of antibodies against PEDV. Molecular assays such as real-time reverse transcriptase-PCR (rRT-PCR) became the methods of choice for the diagnosis of PEDV infection, providing sensitive, specific and rapid detection of the virus RNA in clinical samples. Whereas serological assays have been widely used to monitor prior exposure to the virus and to evaluate the efficacy of novel vaccine candidates or vaccination strategies. Here we discuss the properties of current PEDV diagnostic assays and prospects for improving diagnostic strategies in the future.

Identification of two auto-cleavage products of nonstructural protein 1 (nsp1) in porcine reproductive and respiratory syndrome virus infected cells: nsp1 function as interferon antagonist.

The porcine reproductive and respiratory syndrome virus nsp1 is predicted to be auto-cleaved from the replicase polyprotein into nsp1alpha and nsp1beta subunits. In infected cells, we detected the actual existence of nsp1alpha and nsp1beta. Cleavage sites between nsp1alpha/nsp1beta and nsp1beta/nsp2 were identified by protein microsequencing analysis. Time course study showed that nsp1alpha and nsp1beta mainly localize into the cell nucleus after 10 h post infection. Further analysis revealed that both proteins dramatically inhibited IFN-beta expression. The nsp1beta was observed to significantly inhibit expression from an interferon-stimulated response element promoter after Sendai virus infection or interferon treatment. It was further determined to inhibit nuclear translocation of STAT1 in the JAK-STAT signaling pathway. These results demonstrated that nsp1beta has ability to inhibit both interferon synthesis and signaling, while nsp1alpha alone strongly inhibits interferon synthesis. These findings provide important insights into mechanisms of nsp1 in PRRSV pathogenesis and its impact in vaccine development.

Evaluation of the risk of PRRSV transmission via ingestion of muscle from persistently infected pigs.

The objectives of this experiment were to determine how long porcine reproductive and respiratory syndrome virus (PRRSV) could be detected in muscle tissues of experimentally infected pigs and to evaluate the transmissibility of PRRSV to pigs via ingestion of quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR)-positive muscle tissues. Serum, lymphoid tissues, and muscle (M. longissimus dorsi) samples were collected from 135 pigs (89 PRRSV-inoculated pigs and 46 negative control). Between 28 and 202 days post-inoculation, 13 of 89 (14.6%) muscle samples were positive by qRT-PCR. Among these 13, PRRSV was isolated from four of the 13 corresponding serum samples and three of 13 lymphoid tissue samples. In addition, infectious virus was detected in lymphoid tissue homogenates of six of 13 pigs by intramuscular bioassay. Swine transmissibility studies were performed by feeding thirteen 3-week-old PRRSV-naive pigs (recipient pigs) qRT-PCR-positive muscle and then monitoring recipients for evidence of PRRSV viremia by qRT-PCR. No transmission of PRRSV to recipient pigs via consumption of muscle samples was observed. These data suggested that qRT-PCR detected non-infectious PRRSV in pig meat and/or PRRSV is not highly transmissible to susceptible pigs via consumption of PRRSV-contaminated meat.

Immune response against porcine reproductive and respiratory syndrome virus during acute and chronic infection.

A significant obstacle to the prevention and control of porcine reproductive and respiratory syndrome virus (PRRSV) is the inability of current diagnostic tests to provide information concerning the stage of PRRSV infection. To explore possible prognostic combinations of cell-mediated and humoral immune responses, 3-week-old pigs (n=10) were intramuscularly (IM) inoculated with PRRSV isolate VR-2332 and followed for 193 days post-inoculation (DPI). Negative control pigs (n=10) were IM inoculated with minimum essential medium (MEM). At approximately 2-week intervals, blood samples were collected from all animals and tested for the number of interferon (IFN)-gamma-secreting peripheral blood mononuclear cells (enzyme-linked immunosorbent spot, Elispot), PRRSV viremia (quantitative reverse-transcriptase polymerase chain reaction, qRT-PCR), and serum antibodies using PRRSV protein ELISAs (N, GP5 3', GP5 5', M 5', M 3', GP5-M, and nsp2p) and a commercial PRRSV ELISA (IDEXX Laboratories Inc.). All pigs were viremic by 7 days post-inoculation, with 50% of the pigs resolving viremia by 56 DPI. A PRRSV-specific IFN-gamma response was detected at DPI 28, reached a plateau at 42 DPI, declined slightly, and remained relatively stable from 56 to 193 DPI. On the basis of ROC area under the curve (AUC) analysis, the ELISAs that most reliably differentiated PRRSV-inoculated pigs from negative control pigs were the commercial ELISA (AUC=0.97), the N ELISA (AUC=0.96), and the M 3' ELISA (AUC=0.93). Multivariate analyses were performed to evaluate the relationship between the immune response and the duration and level of viremia. With all antibody assays and Elispot included in the models, the analysis determined that the serum-virus neutralizing antibody response was the best predictor of both level and duration of viremia. It was concluded that humoral antibody responses, particularly the commercial ELISA, N ELISA, and M 3' ELISA were good predictors of prior exposure to PRRSV, but provided little information regarding the ontogeny of the protective immune response. Likewise, cell-mediated immunity based on the number of IFN-gamma-secreting lymphocytes was a poor prognosticator of PRRSV infection status.

Porcine reproductive and respiratory syndrome virus productively infects monocyte-derived dendritic cells and compromises their antigen-presenting ability.

Dendritic cells (DC) are potent antigen-presenting cells that play an important role in inducing primary antigen-specific immune responses. However, some viruses have evolved to specifically target DC to circumvent the host's immune responses for their persistence in the host. Porcine reproductive and respiratory syndrome virus (PRRSV) causes a persistent infection in susceptible animals. Although it is generally believed that the existence of PRRSV quasispecies is partly responsible for the virus persistence, other mechanisms of immune evasion or immune suppression may also exist. Here, we studied the role of DC in PRRSV persistence and immune suppression. Our results showed that PRRSV underwent a productive replication in pig monocyte-derived DC (Mo-DC) as measured by both immunofluorescence staining of viral nucleocapsid protein and virus titration assays, leading to cell death via both apoptosis and necrosis mechanisms. Additionally, PRRSV infection of Mo-DC resulted in reduced expression of MHC class I, MHC class II, CD14 and CD11b/c. This was in agreement with the impaired mixed lymphocyte reaction of PRRSV-infected Mo-DC compared to that of mock-infected Mo-DC. We also examined the cytokine profiles of PRRSV-infected Mo-DC using a quantitative ELISA method. Results indicated that no apparent change in the levels of IL-10, IL-12 and IFN-gamma was detected. Taken together, our data demonstrate that PRRSV productively infects Mo-DC and impairs the normal antigen presentation ability of Mo-DC by inducing cell death, down-regulating the expression of MHC class I, MHC class II, CD11b/c and CD14 and by inducing minimal Th1 cytokines.

Detection of U.S., Lelystad, and European-like porcine reproductive and respiratory syndrome viruses and relative quantitation in boar semen and serum samples by real-time PCR.

Transmission of porcine reproductive and respiratory syndrome virus (PRRSV) via boar semen has been documented. Since semen is widely disseminated for artificial insemination and the virus can cause significant health and economic consequences, it is essential to have well-validated, rapid diagnostic techniques to detect and quantitate the virus for diagnostic and research purposes. Previously, boar semen was tested by a nested PCR (nPCR) assay which was compared to the "gold standard" swine bioassay. A correlation of 94% was observed, indicating that, most of the time, PCR detected infectious virus. Subsequently, a real-time PCR targeting the 3' untranslated region of the PRRSV genome was compared with nPCR by testing 413 serum and semen samples from PRRSV-inoculated and control boars. There was 95% agreement between the results of the two tests, with the majority of samples with discordant results containing virus at the lower range of detection by the assays. The virus in all samples was quantitated by using a standard curve obtained by serial dilution of an in vitro transcript. By using the in vitro transcript, the lower limit of sensitivity was observed to be approximately 33 copies/ml. Reactivity with a panel of more than 100 PRRSV isolates from various geographical regions in the United States was also documented. No reactivity with nine nonrelated swine viruses was noted. A real-time PCR was also developed for the detection of the European Lelystad virus and the European-like PRRSV now found in the United States. In six of six PRRSV-inoculated boars, peak levels of viremia occurred at 5 days postinoculation (DPI) and were most consistently detectable throughout 22 DPI. In five of six boars, PRRSV was shed in semen for 0 to 2 days during the first 10 DPI; however, one of six boars shed the virus in semen through 32 DPI. Therefore, in general, the concentration and duration of PRRSV shedding in semen did not correlate with the quantity or duration of virus in serum. These differences warrant further studies into the factors that prevent viral replication in the reproductive tract.

A 10-kDa structural protein of porcine reproductive and respiratory syndrome virus encoded by ORF2b.

The major structural proteins of porcine reproductive and respiratory syndrome virus (PRRSV) are derived from ORFs 5, 6, and 7. Western blots of sucrose gradient-purified virions and PRRSV-infected MARC-145 cells, probed with immune pig serum, showed the presence of an additional 10-kDa protein. Nucleotide sequence analysis of North American PRRSV isolate SDSU-23983 revealed a small ORF within ORF2, named ORF2b, which, when translated, produced a 73-amino-acid nonglycosylated protein. Recombinant 2b protein expressed by a baculovirus clone, AcVR2, comigrated with the 10-kDa virus-associated protein. The loss of 10-kDa protein immunoreactivity after absorption of immune sera with lysates from AcVR2-infected insect cells demonstrated that the 2b and 10-kDa proteins are immunologically similar. Immunoblots were also used for the detection of anti-2b activity in serum samples from experimentally infected adult pigs. Antibodies against PRRSV were apparent by 14 days postinfection, followed by anti-2b activity and serum neutralizing activity. The putative ORF2b start codon is only 6 nucleotides downstream of the adenine of the ORF2a start codon. The expression of ORF2a and 2b as enhanced green fluorescent fusion proteins showed that both proteins were translated; however, the ORF2b was preferentially expressed. These results suggest that the 2b protein is virion associated and the principal product of ORF2.

Detection and duration of porcine reproductive and respiratory syndrome virus in semen, serum, peripheral blood mononuclear cells, and tissues from Yorkshire, Hampshire, and Landrace boars.

Because transmission of porcine reproductive and respiratory syndrome virus (PRRSV) can occur through boar semen, it is important to identify persistently infected boars. However, even for boars given the same PRRSV strain and dose, variability in the duration of viral shedding in semen has been observed, suggesting that host factors are involved in PRRSV persistence. To determine whether there are host genetic factors, particularly litter and breed differences related to the persistence of PRRSV, 3 litters from 3 purebred swine breeds were used for this study. It was also determined whether PRRSV could be detected for a longer period of time in serum, semen, or peripheral blood mononuclear cells (PBMC) and if PRRSV could still be detected in tissues after these antemortem specimens were PRRSV negative for a minimum of 2-3 weeks. Three Hampshire, 3 Yorkshire, and 2 Landrace PRRSV-naive boars were obtained and inoculated intranasally with a wild-type PRRSV isolate (SD-23983). All boars within each breed were from the same litter, and litters were within 9 days of age. Serum and PBMC were collected twice weekly from each boar and analyzed for the presence of PRRSV by virus isolation and the polymerase chain reaction (PCR). Serum was also used to obtain virus neutralization titers and enzyme-linked immunosorbent assay S/P values. Semen was collected twice weekly from 7 of 8 boars and analyzed by PCR. After all specimens were PRRSV negative for a minimum of 2-3 weeks, each boar was euthanized, and 21 tissues plus saliva, serum, feces, and urine were collected. All postmortem specimens were evaluated by virus isolation. Specimens that were PRRSV negative by virus isolation were then evaluated by PCR. The mean number of days (+/-SD) for the duration of PRRSV shedding in semen was 51+/-26.9 days, 7.5+/-4.9 days, and 28.3+/-17.5 days for Landrace, Yorkshire, and Hampshire boars, respectively. Because of small sample sizes and large SDs, the differences in duration of PRRSV shedding in semen between breeds were not considered significant. However, the trend suggested that Yorkshire boars were more resistant to PRRSV shedding in semen than were Landrace boars, requiring further investigation using a larger numbers of boars. PRRSV was detected for a longer period in semen than in serum or PBMC in 4 of 7 boars. Viremia could be detected for a longer period in serum than in PBMC in 6 of 8 boars. After a minimum of 2-3 weeks of PRRSV-negative serum, semen, and PBMC, PRRSV could still be detected in the tonsil of 3 of 8 boars by virus isolation, indicating that boars still harbor PRRSV within the tonsil even though antemortem specimens are PRRSV negative.

Measurement of immunoglobulin G, A and M concentrations in boar seminal plasma.

How the immune system relates to the boar reproductive tract is not well defined. This is an important area of study because disease-causing agents may be transmitted through boar semen. We have previously identified porcine reproductive and respiratory syndrome virus (PRRSV) in boar semen and wanted to identify PRRSV-specific antibodies within seminal plasma. However, literature documenting total immunoglobulin concentration or the predominant immunoglobulin isotype in boar semen was not available. Therefore, we developed a sandwich enzyme-linked immunoassay (ELISA) to quantitate total IgG, IgA and IgM in seminal plasma from 16 healthy, nonvaccinated, adult boars (n = 102 semen samples). In seminal plasma, IgG was the predominant isotype followed by IgA and IgM. Mean levels +/- the standard deviation followed by the 95% confidence interval of IgG, IgA and IgM were 23.2 +/- 14 microg/mL (15.5 to 31.0), 4.8 +/- 2.5 microg/mL (3.5 to 6.2) and 3.7 +/- 1.7 microg/mL (2.7 to 4.7), respectively. These concentrations of immunoglobulins in seminal plasma were considerably lower than in other swine secretions, which might allow for the survival of infectious agents in boar semen.

Identification of porcine reproductive and respiratory syndrome virus in semen and tissues from vasectomized and nonvasectomized boars.

Previous studies have indicated that porcine reproductive and respiratory syndrome virus (PRRSV) can be identified in and transmitted through boar semen. However, the site(s) of replication indicating the origin of PRRSV in semen has not been identified. To determine how PRRSV enters boar semen, five vasectomized and two nonvasectomized PRRSV-seronegative boars were intranasally inoculated with PRRSV isolate VR-2332. Semen was collected three times weekly from each boar and separated into cellular and cell-free (seminal plasma) fractions. Both fractions were evaluated by reverse transcriptase nested polymerase chain reaction (RT-nPCR) for the presence of PRRSV RNA. Viremia and serostatus were evaluated once weekly, and boars were euthanatized 21 days postinoculation (DPI). Tissues were collected and evaluated by RT-nPCR, virus isolation (VI), and immunohistochemistry to identify PRRSV RNA, infectious virus, or viral antigen, respectively. PRRSV RNA was identified in semen from all vasectomized and nonvasectomized boars and was most consistently found in the cell fraction, within cells identified with a macrophage marker. Viral replication as determined by VI was predominately found within lymphoid tissue. However, PRRSV RNA was widely disseminated throughout many tissues, including the reproductive tract at 21 DPI. These results indicate that PRRSV can enter semen independent of testicular or epididymal tissues, and the source of PRRSV in semen is virus-infected monocytes/macrophages or non-cell-associated virus in serum. PRRSV-infected macrophages in semen may result from infection of local tissue macrophages or may originate from PRRSV-infected circulating monocytes or macrophages.

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.

Effects of a modified-live virus vaccine against porcine reproductive and respiratory syndrome in boars.

OBJECTIVES: To determine whether vaccine virus is found in serum and semen of vaccinated boars, whether vaccination prevents subsequent shedding of wild-type virus after challenge exposure, and whether semen and blood variables are altered after vaccination or challenge exposure with wild-type virus, or both. DESIGN: Throughout the 50-day postvaccination period, serum and semen from exposed boars were evaluated for the presence of porcine reproductive and respiratory syndrome virus (PRRSV). All boars were then challenge-exposed with PRRSV isolate VR-2332 and evaluated for an additional 27 days. Semen quality variables, serostatus, and blood variables were monitored. ANIMALS: 7 PRRSV-seronegative adult boars. PROCEDURE: Semen was collected 3 times weekly and evaluated by use of a nested reverse-transcriptase polymerase chain reaction for detection of PRRSV RNA. Serum was obtained weekly and evaluated by nested reverse-transcriptase polymerase chain reaction, virus isolation, and PRRSV ELISA. Semen quality variables were evaluated 3 times weekly, and CBC was performed weekly. RESULTS: Vaccine virus was shed in the semen of all vaccinated boars, but shedding was of shorter duration in 4 of 5 vaccinated boars than that generally observed after exposure to wild-type virus. After challenge exposure, shedding of wild-type virus in semen was shortened or eliminated in 4 of 5 vaccinated boars. Percentage of forward movement and normal spermatozoal morphology and motility were significantly reduced in vaccinated boars after challenge exposure. CONCLUSIONS: Vaccine virus was shed in semen of vaccinated boars, but vaccination generally reduced or eliminated shedding of wild-type PRRSV after challenge exposure. Semen quality appeared to be less than optimal, particularly after vaccination and subsequent challenge exposure with wild-type virus. CLINICAL RELEVANCE: Extra-label use of the PRRSV vaccine in boars remains controversial because some boars may still shed wild-type virus in semen after challenge exposure at postvaccination day 50. Semen quality also appeared to be altered after vaccination and subsequent challenge exposure.

Chronological immunohistochemical detection and localization of porcine reproductive and respiratory syndrome virus in gnotobiotic pigs.

An immunogold-silver immunohistochemical technique was used to determine the chronological distribution and localization of porcine reproductive and respiratory syndrome virus (PRRSV) in experimentally infected gnotobiotic pigs. Thirty-two pigs were randomly allocated to infected (n = 24) or control (n = 8) groups. Pigs in infected groups were inoculated at 3 days of age by nasal instillation of PRRSV isolate ATCC VR-2332 (total dose = 10(2.64) TCID50), and control pigs were exposed in the same manner to uninfected cell culture supernatant. Three infected and one control pigs were euthanatized at 12 hours and at 1, 2, 3, 5, 7, 14, and 21 days postexposure (DPE). Bronchiolar epithelial cells, arteriolar endothelial cells, monocytes, and interstitial, alveolar, and intravascular macrophages stained for PRRSV antigen at 12 hours postexposure. Staining for PRRSV antigen in endothelial cells, monocytes, and alveolar, interstitial, and intravascular macrophages was most intense and widespread in lung sections from 14 and 21 DPE. In the heart, macrophages in the interstitial and subendocardial spaces and endothelial cells in a few arterioles stained for PRRSV antigen at 14 and 21 DPE. Tonsillar macrophages and mucosal epithelium stained for PRRSV antigen at 12 hours postexposure and sporadically with less intensity in subsequent sampling periods. In the nasal turbinate, PRRSV antigen was identified in macrophages within the mucosal epithelium at 12 hours postexposure and again at 14 and 21 DPE. There was focal staining for PRRSV antigen in the choroid plexus in one pig at 14 DPE. Based on the results of this experiment, the pathogenesis of PRRSV infection in gnotobiotic pigs can be described as initial virus entry through nasal epithelial, tonsillar, and pulmonary macrophages, with viremia occurring by 12 hours postexposure followed by the development of pneumonia, myocarditis, encephalitis, rhinitis, vasculitis, and lymphoid necrosis. Although PRRSV can infect macrophages in heart, tonsil, turbinate, and choroid plexus, pulmonary macrophages are predominantly and consistently infected and are the predominantly cells for virus replication in gnotobiotic pigs.

Persistence of porcine reproductive and respiratory syndrome virus in serum and semen of adult boars.

Four seronegative adult boars were intranasally inoculated with porcine reproductive and respiratory syndrome virus (PRRSV) isolate VR-2332. Serum and semen were collected 2-3 times weekly for over 100 days postinoculation (DPI). Serum samples were assayed for PRRSV by virus isolation (VI) and a polymerase chain reaction (PCR) and screened for antibodies to PRRSV using the indirect fluorescent antibody (IFA) and virus neutralization (VN) tests. Semen was assayed for PRRSV RNA by PCR. Virus and viral RNA was detected in the serum of all boars within 1 DPI by Vi and/or PCR. However, VI results indicated that viremia was transient and occurred from 1 to 9 DPI. Viral RNA was detected in serum from 1 to 31 DPI. In the acute stage of the infection, PRRSV RNA was detected in serum by PCR prior to the presence of viral RNA in semen. The PRRSV RNA was detected in semen as early as 3 DPI and persisted for 25 DPI in 2 of the boars and 56 and 92 DPI in the remaining 2 boars. Detection of PRRSV RNA in semen occurred 2-8 and 28-35 days prior to the detection of antibodies by IFA and VN, respectively. PRRSV was isolated from the bulbourethral gland of the boar that shed viral RNA in semen for 92 DPI. These results suggest that PRRSV RNA can be detected by PCR in boar serum and semen, and may persist for variable periods of time. Viremia and the serologic status of the boar are not adequate indicators of when PRRSV or PRRSV RNA is being shed in the semen. Preliminary findings also indicated that neither shipping stress nor reinoculation with homologous PRRSV resulted in viremia or viral RNA shedding in semen.

The effect of high fat diet and dehydroepiandrosterone (DHEA) administration in the rhesus monkey.

Dehydroepiandrosterone (DHEA) lowers serum cholesterol, particularly the low density lipoprotein (LDL) fraction, in rhesus monkeys on a commercial diet (12% calories from fat, 0.0083% cholesterol). We fed rhesus monkeys a diet of 30% calories from fat and 0.1% cholesterol for 12 weeks, then commercial chow for 7 weeks. Six monkeys each received DHEA or placebo, orally for 17 weeks. Food intake increased the first 6 weeks, but decreased thereafter. Monkeys had a 22% mean weight gain while on high fat diet. DHEA monkeys had higher T4 levels than placebo monkeys at weeks 8 and 16. After 12 weeks on high fat diet, all monkeys had elevated serum cholesterol concentrations, an increase in amount and percentage of intermediate density lipoprotein and LDL cholesterol, and an increase in amount, but a decrease in percentage of high density lipoprotein cholesterol. There were no significant differences in serum cholesterol or plasma lipoprotein concentrations between DHEA and placebo monkeys while on high fat diet (a trend toward lower levels was noted in the DHEA group). On commercial chow, plasma lipoprotein concentrations decreased for all monkeys, and DHEA monkeys had significantly lower total and LDL cholesterol than placebo monkeys. We conclude that a high fat diet (30% fat) masks any cholesterol-lowering effects of DHEA.

Pathogenesis of porcine reproductive and respiratory syndrome virus infection in gnotobiotic pigs.

The pathogenesis of porcine reproductive and respiratory syndrome virus (PRRSV) was determined in gnotobiotic pigs by studying the sequential development of microscopic lesions and sites of virus distribution and replication. Thirty-two pigs (three pigs/infected group and one pig/control group) were inoculated by nasal instillation of either PRRSV isolate ATCC VR-2332 (total dose 10(2.6) TCID50) or uninfected cell culture supernatant. Infected and control pigs were euthanized at 12 hours, and 1, 2, 3, 5, 7, 14, and 21 days postexposure (PE). Gnotobiotic pigs experimentally infected with PRRSV were viremic by 12 hours PE and subsequently developed pneumonia, lymphadenopathy, vasculitis, myocarditis and encephalitis. Lung lesions developed by day 3 PE, persisted through day 21 PE and were characterized by alveolar septa thickened by macrophages, alveolar proteinaceous and karyorrhectic debris, alveolar syncytial cells, and multifocal type II pneumocyte hypertrophy. Lymph node lesions varied in distribution and severity and were characterized by germinal center hypertrophy and hyperplasia, lymphocyte necrosis, multiple cystic spaces, and polykaryocytes within the cystic spaces. Heart lesions were a late feature of infection and all infected pigs had heart lesions on day 21 PE characterized by subendocardial, myocardial, and perivascular foci of lymphocytes. Vasculitis also varied in distribution and severity and affected all sizes of vessels. Results of this experiment indicate that PRRSV is a multisystem disease characterized initially by viremia with subsequent virus distribution and replication in multiple organs causing interstitial pneumonia, vasculitis, lymphadenopathy, myocarditis, and encephalitis.