David A. Benfield

Characterization of Swine Infertility and Respiratory Syndrome (SIRS) Virus (Isolate ATCC VR-2332)

The characterization of an isolate of swine infertility and respiratory syndrome (SIRS) virus (ATCC VR-2332) is reported. A commercial cell line (CL262 1) was used for the propagation of the virus for all assays. Laboratory studies indicate that this isolate is a fastidious, nonhemagglutinating, enveloped RNA virus. Cesium chloride-purified virions visualized by electron microscopy were spherical particles with an average diameter of 62 nm (range: 48–83 nm) and a 25–30 nm core surrounded by an envelope. Virus replication was restricted to the cytoplasm, as demonstrated by immunofluorescence. The virus did not react serologically with antisera to several common porcine viruses or with antisera to known viruses in the alphavirus, rubivirus, pestivirus, and ungrouped lactic dehydrogenase virus genera of the Togaviridae. However, convalescent sow sera and rabbit hyperimmune sera neutralized the SIRS virus at titers of 1:256 and 1:512, respectively. The virus was stable at 4 and −70 C, but was labile at 37 and 56 C. The properties of this isolate of SIRS virus resemble those of the family Togaviridae but do not match the described genera.

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 102,6 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.

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 or 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.

Serum immune responses to the proteins of porcine reproductive and respiratory syndrome (PRRS) virus.

The antibody responses of pigs to porcine reproductive and respiratory syndrome virus (isolate VR-2332) were evaluated by indirect immunofluorescence, virus neutralization, and immunoblotting. All pigs in each group were positive by indirect immunofluorescence 14-21 days postexposure (DPE), and antibodies to specific viral proteins (15, 19 or 26 kD) were initially demonstrated by immunoblotting at 7–21 days DPE. Neutralizing antibodies were detected in only 2 pigs that were inoculated intranasally and given additional parenteral injections with adjuvant. These antibodies appeared much later, 51–70 DPE, than did antibodies detected by indirect immunofluorescence. The titer of the neutralizing antibodies increased until 127 DPE, after which the titers decreased, and 1 animal became seronegative for neutralizing antibody by 262 DPE.

The localization of porcine reproductive and respiratory syndrome virus nucleocapsid protein to the nucleolus of infected cells and identification of a potential nucleolar localization signal sequence

The nucleocapsid (N) protein of porcine reproductive and respiratory syndrome virus (PRRSV) possesses two regions in the N-terminal half of the protein that are enriched in basic amino acids. Presumably, these basic regions are important for packaging the RNA genome within the nucleocapsid of the virus. The PSORT computer program identified the same regions as nuclear localization signal (NLS) sequence motifs. N protein localization to the nucleus of infected MARC-145 and porcine pulmonary macrophages was observed following staining with SDOW-17 and SR-30 anti-N monoclonal antibodies. Furthermore, the co-localization of SR-30 antibody with human ANA-N autoimmune serum identified the nucleolus as the primary site for N protein localization within the nucleus. The localization of the N protein in the absence of infection was studied by following fluorescence in MARC-145 cells transfected with a plasmid, which expressed the nucleocapsid protein fused to an enhanced green fluorescent protein (N-EGFP). Similar to infected cells, N-EGFP localized to the cytoplasm and the nucleolus. Results following the transfection of cells with pEGFP fused to truncated portions of the N gene identified a region containing the second basic stretch of amino acids as the nucleolar localization signal (NoLS) sequence. Another outcome following transfection was the rapid disappearance of cells that expressed high levels of N-EGFP. However, cell death did not correlate with localization of N-EGFP to the nucleolus.

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 = 102.64 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 predominant cells for virus replication in gnotobiotic pigs.

Inhibition of porcine reproductive and respiratory syndrome virus by interferon-gamma and recovery of virus replication with 2-aminopurine

Summary. Porcine reproductive and respiratory syndrome virus (PRRSV) belongs to a group of RNA viruses that establish persistent infections. A proposed strategy for evading immunity during persistent PRRSV infection is by preventing the induction of IFN activity in pigs and/or by blocking the activation of antiviral proteins in permissive cells. IFN-γ mRNA expression was observed in the lymph nodes and lungs of pigs infected with wild-type PRRSV strain SDSU-23983. Pretreatment of MARC-145 cells with IFN-γ inhibited wild-type (SDSU-23983 P6) and culture-adapted (SDSU-23983 P136) PRRS viruses in a dose-dependent manner and at relatively low concentrations. The effect of IFN-γ on virus replication included reductions in the number of infected cells, virus yield, and RNA content in single cells. Virus replication was partially restored by the addition of 2-aminopurine (2-AP), an inhibitor of dsRNA inducible protein kinase (PKR). The addition of 2-AP also restored the viral RNA content per cell to near normal levels, suggesting that inhibition of viral RNA synthesis was through PKR. The principal difference between P6 and P136 isolates was the recovery of P136 replication with lower concentrations of 2-AP. Immunostaining with anti-PKR antibody showed a redistribution of PKR from the cytoplasm into nucleoli of infected cells.

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-naïve 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.

Characterization of homotypic and heterotypic VP7 neutralization sites of rhesus rotavirus

The gene 9 nucleotide sequence was determined for rhesus rotavirus and each of 14 viral variants selected for their resistance to neutralizing monoclonal antibodies. Each variant contains a single gene 9, VP7, mutation which permits viral growth in the presence of the antibody. Variant mutations were identified in two distinct neutralization regions. Region A was identified by monoclonal antibodies that are involved in both serotype-specific and serotype cross-reactive neutralization. Region C was identified by serotype-specific neutralizing monoclonal antibodies. Heterotypic neutralizing monoclonal antibody 57-8 selected variants with a mutation at amino acid 94 in the A region, the same amino acid location selected by serotype-specific monoclonal antibodies. Monoclonal antibody 3 selected a VP7 mutation at amino acid 99 resulting in additional N-linked glycosylation of the VP7 protein. Despite the added VP7 glycosylation, variant v3 was not broadly resistant to additional VP7-specific neutralizing monoclonal antibodies.

Lymphoid tissue tropism of porcine reproductive and respiratory syndrome virus replication during persistent infection of pigs originally exposed to virus in utero.

Abstract The ability of porcine reproductive and respiratory syndrome virus (PRRSV) to establish a persistent infection is the principal contributing factor to the world-wide spread of the disease. Several studies have documented the course of viral infection in postnatally infected pigs; however, very little is known regarding sites of virus replication during persistent infection of pigs exposed to PRRSV in utero. In this study, virus replication and PRRSV-specific antibody were followed for several hundred days in a group of pigs derived from three sows infected at 90 days of gestation with PRRSV isolate VR-2332. Eighty-four percent of pigs were born viremic with a mortality of 54% within 21 days after birth. At approximately 60 days sera from pigs were negative for virus by virus isolation. Analysis of virus replication in the tissues of pigs randomly sacrificed between 63 and 132 days showed no evidence of virus in lung and other non-lymphoid organs. However, virus was easily recovered from tonsil and lymph nodes and in situ hybridization identified these tissues as sites of virus replication. Even though replication was at a low level, virus was easily transmitted to sentinel pigs. By 260 days pigs became seronegative and did not transmit virus to sentinel pigs. Sacrifice of remaining pigs after 300 days showed no evidence of virus in blood and tissues. This study shows that congenital PRRSV-infected pigs can support virus replication for an extended period during which virus replication is primarily restricted to tonsil and lymph nodes.