Equine arteritis virus does not induce interferon production in equine endothelial cells: identification of nonstructural protein 1 as a main interferon antagonist.

The objective of this study was to investigate the effect of equine arteritis virus (EAV) on type I interferon (IFN) production. Equine endothelial cells (EECs) were infected with the virulent Bucyrus strain (VBS) of EAV and expression of IFN-ß was measured at mRNA and protein levels by quantitative real-time RT-PCR and IFN bioassay using vesicular stomatitis virus expressing the green fluorescence protein (VSV-GFP), respectively. Quantitative RT-PCR results showed that IFN-ß mRNA levels in EECs infected with EAV VBS were not increased compared to those in mock-infected cells. Consistent with quantitative RT-PCR, Sendai virus- (SeV-) induced type I IFN production was inhibited by EAV infection. Using an IFN-ß promoter-luciferase reporter assay, we subsequently demonstrated that EAV nsps 1, 2, and 11 had the capability to inhibit type I IFN activation. Of these three nsps, nsp1 exhibited the strongest inhibitory effect. Taken together, these data demonstrate that EAV has the ability to suppress the type I IFN production in EECs and nsp1 may play a critical role to subvert the equine innate immune response.

An autophagy-independent role for LC3 in equine arteritis virus replication.

Equine arteritis virus (EAV) is an enveloped, positive-strand RNA virus. Genome replication of EAV has been associated with modified intracellular membranes that are shaped into double-membrane vesicles (DMVs). We showed by immuno-electron microscopy that the DMVs induced in EAV-infected cells contain double-strand (ds)RNA molecules, presumed RNA replication intermediates, and are decorated with the autophagy marker protein microtubule-associated protein 1 light chain 3 (LC3). Replication of EAV, however, was not affected in autophagy-deficient cells lacking autophagy-related protein 7 (ATG7). Nevertheless, colocalization of DMVs and LC3 was still observed in these knockout cells, which only contain the nonlipidated form of LC3. Although autophagy is not required, depletion of LC3 markedly reduced the replication of EAV. EAV replication could be fully restored in these cells by expression of a nonlipidated form of LC3. These findings demonstrate an autophagy-independent role for LC3 in EAV replication. Together with the observation that EAV-induced DMVs are also positive for ER degradation-enhancing α-mannosidase-like 1 (EDEM1), our data suggested that this virus, similarly to the distantly-related mouse hepatitis coronavirus, hijacks the ER-derived membranes of EDEMosomes to ensure its efficient replication.

Evidence that in vitro susceptibility of CD3+ T lymphocytes to equine arteritis virus infection reflects genetic predisposition of naturally infected stallions to become carriers of the virus.

We investigated the correlation between in vitro susceptibility of CD3(+) T lymphocytes to equine arteritis virus (EAV) infection and establishment of persistent infection among 14 stallions following natural infections. The data showed that carrier stallions with a CD3(+) T lymphocyte susceptibility phenotype to in vitro EAV infection may be at higher risk of becoming carriers than those that lack this phenotype (P = 0.0002).

Recent progress in studies of arterivirus- and coronavirus-host interactions.

Animal coronaviruses, such as infectious bronchitis virus (IBV), and arteriviruses, such as porcine reproductive and respiratory syndrome virus (PRRSV), are able to manifest highly contagious infections in their specific native hosts, thereby arising in critical economic damage to animal industries. This review discusses recent progress in studies of virus-host interactions during animal and human coronavirus and arterivirus infections, with emphasis on IBV-host cell interactions. These interactions may be directly involved in viral replication or lead to the alteration of certain signaling pathways, such as cell stress response and innate immunity, to facilitate viral replication and pathogenesis.

Equine arteritis virus is delivered to an acidic compartment of host cells via clathrin-dependent endocytosis.

Equine arteritis virus (EAV) is an enveloped, positive-stranded RNA virus belonging to the family Arteriviridae. Infection by EAV requires the release of the viral genome by fusion with the respective target membrane of the host cell. We have investigated the entry pathway of EAV into Baby Hamster Kidney cells (BHK). Infection of cells assessed by the plaque reduction assay was strongly inhibited by substances which interfere with clathrin-dependent endocytosis and by lysosomotropic compounds. Furthermore, infection of BHK cells was suppressed when clathrin-dependent endocytosis was inhibited by expression of antisense RNA of the clathrin-heavy chain before infection. These results strongly suggest that EAV is taken up via clathrin-dependent endocytosis and is delivered to acidic endosomal compartments.

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% CO(2) in O(2). Three days after LDV infection, ventilation in response to CO(2) 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 degrees 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 CO(2) 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.

Interleukin-6 production by macrophages from BALB/c mice with a chronic infection of lactic dehydrogenase virus.

The production of interleukin-6 (IL-6), which is known as a B cell differentiation factor, by peritoneal macrophages from mice with a chronic lactic dehydrogenase virus (LDV) infection was compared with that from uninfected mice. The same amounts of IL-6 were detected in the culture supernatant of macrophages from LDV-infected mice as those from uninfected mice. Furthermore IL-6 production of macrophages from LDV-infected and uninfected mice was not affected by the addition of indomethacin. These results suggested that many immunological alterations seen in LDV-infected mice may not be due to, at least in part, altered IL-6 production ability of macrophages and the IL-6 production may not be affected by cyclooxygenase-derived products.

Effect of infection by lactic dehydrogenase virus on expression of intercellular adhesion molecule-1 on vascular endothelial cells of pancreatic islets in streptozotocin-induced insulitis of CD-1 mice.

Streptozotocin (SZ)-induced insulitis, which is an animal model for insulin-dependent diabetes mellitus, was suppressed by lactic dehydrogenase virus (LDV) infection. There were no differences in degenerative and necrotic changes of islet cells between SZ-treated mice and SZ-treated mice with LDV infection during the pre-insulitis phase. The degree of insulitis was more severe and the plasma glucose levels were higher in SZ-treated mice than in SZ-treated mice with LDV infection. Severe degenerative and necrotic changes of cells with mononuclear cell infiltration into the islets were seen in the SZ-treated mice. Infiltration of these cells into islets was less in SZ-treated mice with LDV infection. During the pre-insulitis phase, there was slight expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of vascular endothelial cells in and/or around islets in both groups. ICAM-1 expression on vascular endothelial cells increased in parallel to the degree of insulitis. The degree of this expression in SZ-treated mice was higher than in SZ-treated mice with LDV infection. These results suggest that expression of ICAM-1 on vascular endothelial cells in SZ-treated mice may be important for the development of insulitis. Also, decreased expression of ICAM-1 in the islets may be responsible for the inhibition of the development of insulitis seen in SZ-treated mice with LDV infection.

Nitric oxide production by splenic macrophages is not responsible for T cell suppression during acute infection with lactate dehydrogenase-elevating virus.

Cellular immune responses of mice are transiently suppressed during acute infection with lactate dehydrogenase-elevating virus (LDV). Immunosuppression of mice correlated with a greatly impaired in vitro proliferative response of the majority of the T cells to Con A or anti-CD3 Abs, which could not be reversed by the addition of rIL-2. We have examined whether the T cell suppression is caused by nitric oxide (NO) produced by activated macrophages, which are observed in acutely infected mice. Spleen macrophages from 3-day LDV-infected mice exhibited a 6- to 10-fold increased potential for producing NO, measured as nitrite or nitrite plus nitrate in the culture fluid, but produced significant amounts of NO in vitro only when incubated with IFN-gamma produced by Con A-stimulated T cells in the spleen cell population. Furthermore, we found that the concentrations of NO produced by macrophages in cultures of spleen cells from LDV-infected mice in the presence of IFN-gamma were insufficient to cause a reduction in the proliferative response of T cells in the spleen cell population. An excess of activated macrophages had to be added to achieve T cell suppression. NO inhibition of Con A-induced T cell proliferation exhibited a very sharp dose-response curve. In one experiment little suppression was observed at NO concentrations equivalent to 12 microM nitrite and below, whereas almost complete inhibition was observed at twice the NO concentration. We conclude that NO is not responsible for T cell suppression in LDV-infected mice.