Raymond R.R Rowland

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.

Porcine reproductive and respiratory syndrome virus infection of gnotobiotic pigs: sites of virus replication and co-localization with MAC-387 staining at 21 days post-infection

The organ distribution of PRRSV-infected cells in gnotobiotic piglets at 21 days after infection with PRRSV isolate VR-2332 was examined by in situ hybridization. Cells that expressed PRRSV RNA were identified in all tissues examined, including organs not usually characterized as sites of PRRSV infection. PRRSV-infected cells frequently appeared in clusters and were not always associated with microscopic lesions. The expression of PRRSV RNA co-localized with a macrophage monoclonal antibody, MAC-387, in lymph nodes. Some, but not all infected cells stained with MAC-387. The wide distribution of PRRSV-infected cells and co-localization with MAC-387 staining is consistent with the macrophage-tropism of PRRSV and is similar to observations made during persistent infection with other arteriviruses.

Porcine reproductive and respiratory syndrome virus infection in neonatal pigs characterised by marked neurovirulence

Neonatal pigs from three herds of pigs were somnolent and inappetent and had microscopic lesions characterised by severe meningoencephalitis, necrotic interstitial pneumonia and gastric muscular inflammation. Porcine reproductive and respiratory syndrome virus (PRRSV) infection was diagnosed and confirmed by virus isolation, fluorescent antibody examination of frozen lung sections, serology, immunohistochemistry and in situ hybridisation. Each herd had a history of PRRSV infection and was using or had used a modified-live vaccine. The isolates from the affected pigs were genetically distinct from the modified-live vaccine strain of the virus when compared by restriction enzyme analysis and nucleotide sequencing of PRRSV open reading frames 5 and 6. The virus was identified in macrophages or microglia of brain lesions by immunohistochemical staining of brain sections with an anti-PRRsv monoclonal antibody and an anti-macrophage antibody. The replication of the virus in the brain was verified by in situ hybridisation. The meningoencephalitis induced by the virus in pigs from each of the herds was unusually severe and the brain lesions were atypical when compared with other descriptions of encephalitis induced by the virus, which should therefore be considered as a possible diagnosis for neonatal pigs with severe meningoencephalitis. In addition, field isolates of the virus which are capable of causing disease can emerge and coexist with modified-live vaccine virus in some pig herds.

Neuropathogenicity and Susceptibility to Immune Response are Interdependent Properties of Lactate Dehydrogenase-Elevating Virus (LDV) and Correlate with the Number of N-Linked Polylactosaminoglycan Chains on the Ectodomain of the Primary Envelope Glycoprotein

We have developed differential RT-PCR methods to distinguish different isolates of LDV and have purified several quasispecies by repeated end point dilution in mice. They fall into two groups, each possessing two or more members. Group A viruses are non-neuropathogenic, highly resistant to in vitro neutralization by antibodies and efficient in establishment of a life-long, persistently viremic infection in mice despite a detectable immune response. Group B viruses, on the other hand, are neuropathogenic, much more sensitive to antibody neutralization and have an impaired ability to establish a high viremia persistent infection in immune competent mice. These properties seem to be interdependent and correlate with the number of N-glycosylation sites on the short (about 30 amino acid long) ectodomain of the primary envelope glycoprotein, VP-3P, which probably is part of the attachment site for the LDV receptor on permissive cells and harbors an epitope(s) reacting with neutralizing antibodies. Group A viruses possess three closely spaced N-linked polylactosaminoglycan chains, whereas group B viruses lack the two N-terminal ones. We postulate that lack of these polylactosaminoglycan chains endows group B viruses with the ability to interact with a receptor on anterior horn neurons resulting in neuropathogenesis. At the same time, it increases an interaction with neutralizing antibodies thus impeding the infection of macrophages newly generated during the persistent phase of infection which is essential for the continued rounds of replication of the virus.

Inhibition of virus-induced age-dependent poliomyelitis by interferon-γ

Age-dependent poliomyelitis (ADPM) is a neuroparolytic disease which results from combined infection of susceptible mice with lactate dehydrogenase-elevating virus (LDV) and murine leukemia virus (MuLV). The present study examined the effects of interferon-gamma (IFN-gamma) treatment on the incidence of ADPM, replication of LDV and MuLV and anti-LDV immunity. IFN-gamma treatment of ADPM-susceptible C58/M mice protected them from paralytic disease, but had no detectable effect on the IgG anti-LDV response or LDV viremia. IFN-gamma-mediated protection from ADPM correlated with reduced expression of LDV RNA, but not MuLV RNA, in the spinal cords of C58/M mice. These results confirm that spinal cord LDV replication is the determinant of ADPM and demonstrate that cytokine-mediated inhibition of LDV replication in the central nervous system prevents neuroparalytic disease.

Age-dependent poliomyelitis in mice is associated with respiratory failure and viral replication in the central nervous system and lung

Age-dependent poliomyelitis (ADPM) is a virally induced neuroparalytic disease of mice and a model for amyotrophic lateral sclerosis (ALS). ADPM is triggered in genetically susceptible mice by immunosuppression and infection with lactate dehydrogenase-elevating virus (LDV). Both ADPM and ALS are characterized by progressive degeneration of anterior horn motor neurons, and death in ALS is usually associated with respiratory failure. To assess respiratory function in ADPM, we investigated ventilation in conscious control and LDV-infected C58/J mice breathing air and then 6.5% CO2 in O2. Three days after LDV infection, ventilation in response to CO2 was half of that compared to the uninfected state, but become normalized by 10 days. Administration of cyclophosphamide alone (200 mg/kg, ip), an immunosuppressant, had no effect on ventilation. Induction of ADPM by concomitant administration of LDV to cyclophosphamide-treated mice resulted in altered gait, hindlimb paralysis, wasting, decreased metabolism, and decreased body temperature by 4°C relative to controls. Compared to baseline values, mice with ADPM had decreased tidal volume and ventilation while breathing air, and while exposed to the CO2 challenge they were unable to increase tidal volume, frequency of breathing, or ventilation. Using in situ hybridization, LDV replication was noted within the spinal cord, brain, and lung, but not in the diaphragm. Thus, respiratory failure is a contributory mechanism leading to death in ADPM and is associated with LDV replication in the CNS and lung. This animal model may be useful to investigate physiological and molecular mechanisms associated with the development of respiratory failure in neurodegenerative diseases.