Specific Detection of Two Divergent Simian Arteriviruses Using RNAscope In Situ Hybridization.

Simian hemorrhagic fever (SHF) is an often lethal disease of Asian macaques. Simian hemorrhagic fever virus (SHFV) is one of at least three distinct simian arteriviruses that can cause SHF, but pathogenesis studies using modern methods have been scarce. Even seemingly straightforward studies, such as examining viral tissue and cell tropism in vivo, have been difficult to conduct due to the absence of standardized SHFV-specific reagents. Here we report the establishment of an in situ hybridization assay for the detection of SHFV and distantly related Kibale red colobus virus 1 (KRCV-1) RNA in cell culture. In addition, we detected SHFV RNA in formalin-fixed, paraffin-embedded tissues from an infected rhesus monkey (Macaca mulatta). The assay is easily performed and can clearly distinguish between SHFV and KRCV-1. Thus, if further developed, this assay may be useful during future studies evaluating the mechanisms by which a simian arterivirus with a restricted cell tropism can cause a lethal nonhuman primate disease similar in clinical presentation to human viral hemorrhagic fevers.

A simian hemorrhagic fever virus isolate from persistently infected baboons efficiently induces hemorrhagic fever disease in Japanese macaques.

Simian hemorrhagic fever virus is an arterivirus that naturally infects species of African nonhuman primates causing acute or persistent asymptomatic infections. Although it was previously estimated that 1% of baboons are SHFV-positive, more than 10% of wild-caught and captive-bred baboons tested were SHFV positive and the infections persisted for more than 10 years with detectable virus in the blood (100-1000 genomes/ml). The sequences of two baboon SHFV isolates that were amplified by a single passage in primary macaque macrophages had a high degree of identity to each other as well as to the genome of SHFV-LVR, a laboratory strain isolated in the 1960s. Infection of Japanese macaques with 100PFU of a baboon isolate consistently produced high level viremia, pro-inflammatory cytokines, elevated tissue factor levels and clinical signs indicating coagulation defects. The baboon virus isolate provides a reliable BSL2 model of viral hemorrhagic fever disease in macaques.

The recent European isolate (08P178) of equine arteritis virus causes inflammation but not arteritis in experimentally infected ponies.

In the last two decades, outbreaks of equine viral arteritis (EVA) have been reported in Europe, but little is known about these European isolates of equine arteritis virus (EAV). EAV European strain (08P178, EU-1 clade) isolated from one of these recent outbreaks is able to cause clinical signs on experimental infection. The aim of the present study was to investigate the microscopical lesions induced by this isolate after experimental infection of ponies. Animals were killed at 3, 7, 14 and 28 days post infection (dpi). At 3 dpi, lesions were essentially restricted to the respiratory tract and intestines and were characterized by mild multifocal epithelial degeneration and associated mononuclear cell infiltration. Lesions were more severe at 7 dpi and by 14 dpi, respiratory lesions were even more severe and lymphoplasmacytic infiltrates extended to other organs. At 28 dpi, lesions were still present in the viscera. In all specimens the most prominent histological change was intraepithelial, subepithelial and perivascular lymphoplasmacytic infiltration, ranging from mild and multifocal to extensive and diffuse. No signs of arterial damage such as infarcts, haemorrhages or necrosis were found. In conclusion, infection of naïve animals with the European 08P178 strain of EAV is associated with inflammation, but not arteritis.

Identification of target cells of a European equine arteritis virus strain in experimentally infected ponies.

Currently, little is known on the cellular pathogenesis of equine arteritis virus (EAV). The purpose of the present study was to identify the target cells in ponies experimentally inoculated with EAV 08P178 (EU, clade-1). EAV-target organs (respiratory tissues with associated lymphoid tissues and large intestines), collected at 3 and 7 days post inoculation (dpi) and with virus titers≥10(5.0) TCID50/g, were processed with double immunofluorescence staining for the simultaneous detection of EAV N-protein and one of the following cell markers: CD172a (myeloid cells), CD3 (T lymphocytes), IgM (B lymphocytes) and von Willebrand factor (endothelial cells). In the different analyzed organs, 31-58% and 47-63% of the EAV-positive cells were mononuclear leukocytes (mainly CD172a(+) followed by CD3(+)) at 3 and 7 dpi, respectively. EAV-positive endothelial cells were not detected in 3.200 large blood vessels (≥3 endothelial cells/vessel cross section). However, in terminal capillaries (1-2 endothelial cells/vessel cross section) of the different organs, 15-51% of the endothelial cells were EAV-positive. In conclusion, the present study demonstrates that EAV 08P178 (i) has a main tropism for CD172a(+) and CD3(+) mononuclear leukocytes and (ii) infects a large number of endothelial cells in terminal capillaries. EAV 08P178 infection in capillaries is most probably the cause of an increased vascular permeability leading to leakage of fluid (edema-serous exudate) but not to severe vasculitis and hemorrhages.

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.

Regulatory T cells in arterivirus and coronavirus infections: do they protect against disease or enhance it?

Regulatory T cells (T(regs)) are a subset of T cells that are responsible for maintaining peripheral immune tolerance and homeostasis. The hallmark of T(regs) is the expression of the forkhead box P3 (FoxP3) transcription factor. Natural regulatory T cells (nT(regs)) are a distinct population of T cells that express CD4 and FoxP3. nTregs develop in the thymus and function in maintaining peripheral immune tolerance. Other CD4(+), CD4(-)CD8(-), and CD8(+)CD28(-) T cells can be induced to acquire regulatory function by antigenic stimulation, depending on the cytokine milieu. Inducible (or adaptive) T(regs) frequently express high levels of the interleukin 2 receptor (CD25). Atypical T(regs) express FoxP3 and CD4 but have no surface expression of CD25. Type 1 regulatory T cells (Tr1 cells) produce IL-10, while T helper 3 cells (Th3) produce TGF-ß. The function of inducible T(regs) is presumably to maintain immune homeostasis, especially in the context of chronic inflammation or infection. Induction of T(regs) in coronaviral infections protects against the more severe forms of the disease attributable to the host response. However, arteriviruses have exploited these T cell subsets as a means to dampen the immune response allowing for viral persistence. T(reg) induction or activation in the pathogenesis of disease has been described in both porcine reproductive and respiratory syndrome virus, lactate dehydrogenase elevating virus, and mouse hepatitis virus. This review discusses the development and biology of regulatory T cells in the context of arteriviral and coronaviral infection.

Clinical and virological outcome of an infection with the Belgian equine arteritis virus strain 08P178.

Equine viral arteritis (EVA) is an infectious disease with variable clinical outcome. Outbreaks, causing important economic losses, are becoming more frequent. Currently, there is a shortage of pathogenesis studies performed with European strains. In the present study, eight seronegative ponies were experimentally inoculated with the Belgian strain of equine arteritis virus (EAV) 08P178 (EU-1 clade) and monitored daily for clinical signs of EVA. Nasopharyngeal swabs, ocular swabs, bronchoalveolar cells and blood were collected for virological and serological testing. Two ponies were euthanized at 3, 7, 14, and 28 days post infection (DPI). After necropsy, specimens were collected for virus titration and immunofluorescence. EVA symptoms such as fever and lymphadenomegaly were evident from 3 to 10 DPI. Virus was isolated in nasal secretions from 2 to 9 DPI and in bronchoalveolar cells from 3 to 7 DPI. A cell-associated viraemia was detected from 3 to 10 DPI. After replication in the respiratory tract and draining lymph nodes, EAV reached secondary target organs (high virus titers in internal organs sampled at 7 DPI). At 14 DPI, virus titers dropped drastically and, at 28 DPI, only tonsils were positive. Immunofluorescence revealed both individual and clustered EAV-infected cells. Antibodies were detected starting from 7 DPI. It can be concluded that the Belgian strain 08P178 is a European mildly virulent subtype. At present, most European EAV strain infections were thought to run a subclinical course. This study is a proof that mildly virulent European EAV strains do exist in the field.

Simian hemorrhagic fever virus infection of rhesus macaques as a model of viral hemorrhagic fever: clinical characterization and risk factors for severe disease.

Simian Hemorrhagic Fever Virus (SHFV) has caused sporadic outbreaks of hemorrhagic fevers in macaques at primate research facilities. SHFV is a BSL-2 pathogen that has not been linked to human disease; as such, investigation of SHFV pathogenesis in non-human primates (NHPs) could serve as a model for hemorrhagic fever viruses such as Ebola, Marburg, and Lassa viruses. Here we describe the pathogenesis of SHFV in rhesus macaques inoculated with doses ranging from 50 PFU to 500,000 PFU. Disease severity was independent of dose with an overall mortality rate of 64% with signs of hemorrhagic fever and multiple organ system involvement. Analyses comparing survivors and non-survivors were performed to identify factors associated with survival revealing differences in the kinetics of viremia, immunosuppression, and regulation of hemostasis. Notable similarities between the pathogenesis of SHFV in NHPs and hemorrhagic fever viruses in humans suggest that SHFV may serve as a suitable model of BSL-4 pathogens.

Zn(2+) inhibits coronavirus and arterivirus RNA polymerase activity in vitro and zinc ionophores block the replication of these viruses in cell culture.

Increasing the intracellular Zn(2+) concentration with zinc-ionophores like pyrithione (PT) can efficiently impair the replication of a variety of RNA viruses, including poliovirus and influenza virus. For some viruses this effect has been attributed to interference with viral polyprotein processing. In this study we demonstrate that the combination of Zn(2+) and PT at low concentrations (2 µM Zn(2+) and 2 µM PT) inhibits the replication of SARS-coronavirus (SARS-CoV) and equine arteritis virus (EAV) in cell culture. The RNA synthesis of these two distantly related nidoviruses is catalyzed by an RNA-dependent RNA polymerase (RdRp), which is the core enzyme of their multiprotein replication and transcription complex (RTC). Using an activity assay for RTCs isolated from cells infected with SARS-CoV or EAV--thus eliminating the need for PT to transport Zn(2+) across the plasma membrane--we show that Zn(2+) efficiently inhibits the RNA-synthesizing activity of the RTCs of both viruses. Enzymatic studies using recombinant RdRps (SARS-CoV nsp12 and EAV nsp9) purified from E. coli subsequently revealed that Zn(2+) directly inhibited the in vitro activity of both nidovirus polymerases. More specifically, Zn(2+) was found to block the initiation step of EAV RNA synthesis, whereas in the case of the SARS-CoV RdRp elongation was inhibited and template binding reduced. By chelating Zn(2+) with MgEDTA, the inhibitory effect of the divalent cation could be reversed, which provides a novel experimental tool for in vitro studies of the molecular details of nidovirus replication and transcription.

Lactate dehydrogenase-elevating virus induces apoptosis in cultured macrophages and in spinal cords of C58 mice coincident with onset of murine amyotrophic lateral sclerosis.

Age-dependent poliomyelitis (ADPM) or murine amyotrophic lateral sclerosis (ALS) is a murine paralytic disease triggered in immunosuppressed genetically-susceptible mice by infection with the arterivirus lactate dehydrogenase-elevating virus (LDV). This disease provides an animal model for ALS, affecting anterior horn neurons and resulting in neuroparalysis 2-3 weeks after LDV infection. We have tested the hypothesis that spinal cord apoptosis is a feature of the LDV-induced murine ALS, since apoptosis is postulated to be a causal factor in human ALS. Gene microarray analyses of spinal cords from paralyzed animals revealed upregulation of several genes associated with apoptosis. Spinal cord apoptosis was investigated further by TUNEL and activated caspase-3 assays, and was observed to emerge concurrent with paralytic symptoms in both neuronal and non-neuronal cells. Caspase-3-dependent apoptosis was also triggered in cultured macrophages by neurovirulent LDV infection. Thus, virus-induced spinal cord apoptosis is a pre-mortem feature of ADPM, which affects both neuronal and support cells, and may contribute to the pathogenesis of this ALS-like 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% 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.

Reduction of serum interferon (IFN)-gamma concentration and lupus development in NZBxNZWF(1)mice by lactic dehydrogenase virus infection.

The complexity of cytokine regulation and the imbalance of helper T (Th)1 and Th2 subsets in systemic lupus erythematosus (SLE) animal models and human SLE are well recognized. In this study in NZBxNZWF(1)mice, the effects of lactic dehydrogenase virus (LDV) infection on the production of interferon (IFN)-gamma in the serum and the development of autoimmune disease were examined. The progress of the disease (the development of glomerulonephritis, formation of glomerular IgG and C3 deposits, increase in the blood urea nitrogen values, and mortality) was parallel with an increase in serum IFN-gamma in uninfected NZBxNZWF(1)mice. These changes were inhibited in LDV-infected NZBxNZWF(1)mice. Our findings suggest that increase in serum IFN-gamma may be associated with the active disease in NZBxNZWF(1)mice.

Diagnosis of equine arteritis virus infection in two horses by using monoclonal antibody immunoperoxidase histochemistry on skin biopsies.

Two 5-year-old grade male horses presented with epiphora, rhinorrhea, conjunctival and nasal mucosal hyperemia, and dorsal and thoracic macropapular rash. Skin biopsies were collected from the affected areas, and serial sections were evaluated following hematoxylin and eosin and immunoperoxidase histochemistry staining by using a murine monoclonal antibody of the immunoglobulin G2A isotype recognizing the 30-kDa membrane protein of equine arteritis virus (EAV). In both horses, lesions consisted of mild to moderate diffuse superficial dermal edema and vasculitis with mild perivascular lymphocytic infiltrates, occasional endothelial hypertrophy, and single-cell necrosis of tunica media myocytes. Immunohistochemically, a few endothelial cells, myocytes, and pericytes containing intracytoplasmic EAV antigen were identified. Immunoperoxidase histochemistry of skin biopsies can be used as an ancillary test for the clinical diagnosis of equine viral arteritis in horses, especially when a cutaneous macropapular rash is evident.

Equine viral arteritis.

Equine viral arteritis (EVA) can cause prominent economic losses for the equine industry. The purpose of this review is to provide the pathologist some familiarity with the clinical history, lesions, pathogenesis, and diagnosis of EVA. EVA is caused by an arterivirus (equine arteritis virus, EAV), and the vascular system is the principal but not unique viral target. EVA has variable presentations, including interstitial pneumonia, panvasculitis with edema, thrombosis and hemorrhage, lymphoid necrosis, renal tubular necrosis, abortion, and inflammation of male accessory genital glands. EAV antigen (EAVAg) can be demonstrated within the cytoplasm of epithelial cells such as alveolar pneumocytes, enterocytes, adrenal cortical cells, trophoblasts, thymus stroma, renal tubular cells, and male accessory genital gland cells. It can be also demonstrated within endothelia, in vascular, myometrial, and cardiac myocytes, macrophages, dendritelike cells of lymphoid organs, and chorionic mesenchymal stromal cells. In young and adult horses, following colonization of macrophages, the virus spreads systemically using circulating monocytes and enters the endothelium and tunica media of blood vessels, histiocytes, and dendritelike cells. Eventually, the virus multiplies within renal tubular cells. Lesions are uncommon in the aborted fetus; if present, they are mild, and EAVAg is frequently not detectable within fetal tissues and placenta. The clinical presentation and lesions of EVA may resemble those of other diseases. Complete pathologic examination associated with immunohistochemistry, virus isolation, and, especially in cases of abortion, serology will guarantee a directed and accurate diagnosis.

Neuropathogenicity and sensitivity to antibody neutralization of lactate dehydrogenase-elevating virus are determined by polylactosaminoglycan chains on the primary envelope glycoprotein.

Common strains of lactate dehydrogenase-elevating virus (LDV, an arterivirus), such as LDV-P and LDV-vx, are highly resistant to antibody neutralization and invariably establish a viremic, persistent, yet asymptomatic, infection in mice. Other LDV strains, LDV-C and LDV-v, have been identified that, in contrast, are highly susceptible to antibody neutralization and are incapable of a high viremic persistent infection, but at the same time have gained the ability to cause paralytic disease in immunosuppressed C58 and AKR mice. Our present results further indicate that these phenotypic differences represent linked properties that correlate with the number of N-glycosylation sites associated with the single neutralization epitope on the short ectodomain of the primary envelope glycoprotein, VP-3P. The VP-3P ectodomains of LDV-P/vx possess three N-glycosylation sites, whereas those of LDV-C/v lack the two N-terminal sites. We have now isolated four independent neutralization escape variants of neuropathogenic LDV-C and LDV-v on the basis of their ability to establish a high viremic persistent infection in mice. The VP-3P ectodomains of all four variants had specifically regained two N-glycosylation sites concomitant with decreased immunogenicity of the neutralization eptitope and decreased sensitivity to antibody neutralization as well as loss of neuropathogenicity.

Lactic dehydrogenase virus infection inhibits allergic eosinophil reaction and IL-5 gene expression in vivo.

The effects of lactic dehydrogenase virus (LDV) infection on allergic eosinophil reaction and IL-5 gene expression were studied. LDV infection suppressed antigen-induced eosinophil recruitment into the peritoneal cavity in sensitized mice. The elevation of IL-5 gene expression in the spleen and mesenteric lymph nodes 6 h after ovalbumin challenge was significantly suppressed in LDV-infected mice compared with uninfected (control) mice. The expression of the interferon-gamma and IL-2 genes in the spleen, but not in mesenteric lymph nodes, was significantly suppressed in LDV-infected mice compared with control mice. The present results suggest, that suppression of IL-5 gene expression by LDV infection may not be mediated by a mutual inhibitory mechanism between Th1 and Th2 cells.

Lactate dehydrogenase-elevating virus variants: cosegregation of neuropathogenicity and impaired capability for high viremic persistent infection.

Neuropathogenic isolates of lactate dehydrogenase virus (LDV) differ from non-neuropathogenic isolates in their unique ability to cause a paralytic disease (age-dependent poliomyelitis, ADPM) in immunosuppressed C58 and AKR mice by cytocidally infecting their anterior horn neurons. We have recently reported that an original neuropathogenic LDV isolate, LDV-C-BR, contained a low level of a coexisting non-neuropathogenic LDV which, in a mixed infection of mice, rapidly outcompeted the former resulting in apparent loss of neuropathogenicity of the reisolated LDV. This correlated with an impaired ability of the neuropathogenic LDV to establish a viremic persistent infection. In the present study we identified the presence of three different quasispecies in another original neuropathogenic LDV by sequence analysis of cDNA clones of ORF 5 (encoding the primary envelope glycoprotein VP-3P) obtained from the isolate. Successful development of differential reverse transcription-polymerase chain reaction assays allowed us to biologically clone all three quasispecies through repeated end point dilutions. Only one of the quasispecies (LDV-v) was neuropathogenic. The other two, LDV-vP (probably the same as LDV-P) and LDV-vx (a novel LDV quasispecies that had not been previously identified), were non-neuropathogenic and found to be the common LDV quasispecies associated with almost all LDVs originally isolated from mice carrying various other transplantable tumors. The neuropathogenic LDV-v became selectively amplified in the spinal cords of paralyzed mice, but possessed an impaired ability to establish a persistent viremic infection and was rapidly out-competed by LDV-vP and LDV-vx in mixed infections, just as reported previously for LDV-C-BR. The results further support our hypothesis that neuropathogenicity and impaired capability for viremic persistence of LDV are determined by the same molecular feature. The only consistent and biologically relevant molecular difference we have observed between neuropathogenic and non-neuropathogenic LDVs is the number of polylactosaminoglycan chains associated with the ectodomain of VP-3P.

Equine viral arteritis in newborn foals: clinical, pathological, serological, microbiological and immunohistochemical observations.

Clinical, pathological, immunohistochemical, serological and microbiological findings are described for 2 geographically and temporally distinct equine arteritis virus (EAV) epidemics in newborn foals. Outbreak A occurred at a commercial Standardbred breeding facility; Outbreak B began in a group of research animals. Clinical signs were severe and primarily referable to the respiratory tract. Fever and leucopenia and/or thrombocytopenia were observed in foals surviving for more than 24 h. The most common gross pathological findings were limited to the respiratory tract. Common histopathological findings included interstitial pneumonia, lymphocytic arteritis and periarteritis with fibrinoid necrosis of the tunica media. Renal tubular necrosis was noted in 2 foals. Immunoperoxidase histochemistry combined with virus isolation was diagnostic in all cases.

Equine arteritis virus: a review of clinical features and management aspects.

Sero-epidemiological surveys have revealed that equine arteritis virus (EAV) is prevalent in most European countries. The virus causes sporadic cases of respiratory disease and abortion in horses, the incidence of which has increased in recent years. Mares and geldings eliminate virus after acute infection, but 30% to 60% of stallions become persistently infected. In these animals, EAV is maintained within the reproductive tract and is shed continuously in the semen. Persistent infection with EAV in stallions has no negative consequences for fertility but mares inseminated with virus-contaminated semen can have an acute infection. These mares shed large amounts of virus in respiratory secretions and urine, leading to lateral spread of the virus to other susceptible horses. Acute infection at later stages of gestation can lead to abortion. Effective control of the spread of EAV infection depends on the identification of virus-shedding stallions. Persistently infected stallions should not be used for breeding or should be bred only to seropositive mares. Mares bred to shedding stallions should be isolated from other animals for a period of 3 weeks following insemination to prevent the lateral spread of EAV.