Pentosan polysulfate sodium prevents functional decline in chikungunya infected mice by modulating growth factor signalling and lymphocyte activation.

Chikungunya virus (CHIKV) is an arthropod-borne virus that causes large outbreaks world-wide leaving millions of people with severe and debilitating arthritis. Interestingly, clinical presentation of CHIKV arthritides have many overlapping features with rheumatoid arthritis including cellular and cytokine pathways that lead to disease development and progression. Currently, there are no specific treatments or vaccines available to treat CHIKV infections therefore advocating the need for the development of novel therapeutic strategies to treat CHIKV rheumatic disease. Herein, we provide an in-depth analysis of an efficacious new treatment for CHIKV arthritis with a semi-synthetic sulphated polysaccharide, Pentosan Polysulfate Sodium (PPS). Mice treated with PPS showed significant functional improvement as measured by grip strength and a reduction in hind limb foot swelling. Histological analysis of the affected joint showed local inflammation was reduced as seen by a decreased number of infiltrating immune cells. Additionally, joint cartilage was protected as demonstrated by increased proteoglycan staining. Using a multiplex-immunoassay system, we also showed that at peak disease, PPS treatment led to a systemic reduction of the chemokines CXCL1, CCL2 (MCP-1), CCL7 (MCP-3) and CCL12 (MCP-5) which may be associated with the reduction in cellular infiltrates. Further characterisation of the local effect of PPS in its action to reduce joint and muscle inflammation was performed using NanoString™ technology. Results showed that PPS altered the local expression of key functional genes characterised for their involvement in growth factor signalling and lymphocyte activation. Overall, this study shows that PPS is a promising treatment for alphaviral arthritis by reducing inflammation and protecting joint integrity.

Pentosan polysulfate sodium for Ross River virus-induced arthralgia: a phase 2a, randomized, double-blind, placebo-controlled study.

BACKGROUND: Alphaviruses, such as Ross River (RRV) and chikungunya virus (CHIKV), cause significant global morbidity, with outbreaks of crippling joint inflammation and pain, leaving patients incapacitated for months to years. With no available vaccine or specific therapeutic for any alphaviral disease, and a growing economic and public health burden, there is a serious need for the development of specific therapies. METHODS: This study evaluated the safety and efficacy of pentosan polysulfate sodium (PPS) in subjects with RRV-induced arthralgia in a double-blind, placebo-controlled trial. Twenty subjects were randomized 2:1 to subcutaneous PPS (2 mg/kg) or placebo (sodium chloride 0.9%) twice weekly for 6 weeks. Safety evaluation included physical examination, concomitant medications, and laboratory findings. Efficacy assessments included change from baseline in joint function (hand grip strength and RAPID3) and quality of life (SF-36) at Days 15, 29, 39 and 81 after treatment initiation. Inflammatory and cartilage degradation biomarkers were exploratory endpoints. RESULTS: PPS was well tolerated, with a similar proportion of subjects reporting at least one treatment-emergent adverse event (TEAE) in the treatment and placebo groups. Injection site reactions were the most common TEAE and occurred more frequently in the PPS group. Dominant hand grip strength and SF-36 scores improved with PPS at all time points assessed, with hand grip strength improvement of 6.99 kg (p = 0.0189) higher than placebo at Day 15. PPS showed significant improvements versus placebo in adjusted mean relative change from baseline for RAPID3 Pain (p = 0.0197) and Total (p = 0.0101) scores at Day 15. At the conclusion of the study overall joint symptoms, assessed by RAPID3, showed near remission in 61.5% of PPS subjects versus 14.3% of placebo subjects. Additionally, PPS treatment improved COMP, CTX-II, CCL1, CXCL12, CXCL16 and CCL17 biomarker levels versus placebo. CONCLUSIONS: Overall, the improvements in strength and joint symptoms warrant further evaluation of PPS as a specific treatment for RRV-induced and other forms of arthritis. TRIAL REGISTRATION: This trial is registered at the Australian New Zealand Clinical Trials Registry # ACTRN12617000893303 .

Decreased Virulence of Ross River Virus Harboring a Mutation in the First Cleavage Site of Nonstructural Polyprotein Is Caused by a Novel Mechanism Leading to Increased Production of Interferon-Inducing RNAs.

Infection with Ross River virus (RRV) causes debilitating polyarthritis and arthralgia in individuals. Alphaviruses are highly sensitive to type I interferon (IFN). Mutations at the conserved P3 position of the cleavage site between nonstructural protein 1 (nsP1) and nsP2 (1/2 site) modulate type I IFN induction for both RRV and Sindbis virus (SINV). We constructed and characterized RRV-T48A534V, a mutant harboring an A534V substitution in the P1 position of the 1/2 site, and compared it to parental RRV-T48 and to RRV-T48A532V, SINVI538 and SINVT538 harboring different substitutions in the same region. A534V substitution resulted in impaired processing of RRV nonstructural polyprotein and in elevated production of replicase-generated pathogen-associated molecular pattern (PAMP) RNAs that induce expression of type I IFN. Both A532V and A534V substitutions affected synthesis of viral RNAs, though the effects of these closely located mutations were drastically different affecting mostly either the viral negative-strand RNA or genomic and subgenomic RNA levels, respectively. Synthesis of PAMP RNAs was also observed for SINV replicase, and it was increased by I538T substitution. In comparison to RRV-T48, RRV-T48A534V was attenuated in vitro and in vivo Interestingly, when type I IFN-deficient cells and type I IFN receptor-deficient mice were infected with RRV-T48 or RRV-T48A534V, differences between these viruses were no longer apparent. Compared to RRV-T48, RRV-T48A534V infection was associated with increased upregulation of type I IFN signaling proteins. We demonstrate novel mechanisms by which the A534V mutation affect viral nonstructural polyprotein processing that can impact PAMP RNA production, type I IFN induction/sensitivity, and disease.IMPORTANCE This study gives further insight into mechanisms of type I IFN modulation by the medically important alphaviruses Ross River virus (RRV) and Sindbis virus (SINV). By characterizing attenuated RRV mutants, the crucial role of amino acid residues in P1 and P3 positions (the first and third amino acid residues preceding the scissile bond) of the cleavage site between nsP1 and nsP2 regions was highlighted. The study uncovers a unique relationship between alphavirus nonstructural polyprotein processing, RNA replication, production of different types of pathogen-associated molecular pattern (PAMP) RNAs, type I IFN induction, and disease pathogenesis. This study also highlights the importance of the host innate immune response in RRV infections. The viral determinants of type I IFN modulation provide potential drug targets for clinical treatment of alphaviral disease and offer new approaches for rational attenuation of alphaviruses for construction of vaccine candidates.

Chondrocytes Contribute to Alphaviral Disease Pathogenesis as a Source of Virus Replication and Soluble Factor Production.

Arthritogenic alphavirus infections often result in debilitating musculoskeletal disorders that affect the joints, muscle, and bone. In order to evaluate the infection profile of primary human skeletal muscle and chondrocyte cells to Ross River virus (RRV) in vitro, cells were infected at a multiplicity of infection (MOI) of 1 over a period of two days. Viral titers were determined by plaque assay and cytokine expression by Bio-Plex® assays using the supernatants harvested. Gene expression studies were conducted using total RNA isolated from cells. Firstly, we show that RRV RNA is detected in chondrocytes from infected mice in vivo. Both human primary skeletal muscle and chondrocyte cells are able to support productive RRV infection in vitro. We also report the production of soluble host factors including the upregulation of heparanase (HPSE) and inflammatory host factors such as interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), RANTES (regulated on activation, normal T cell expressed and secreted), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), which are also present during clinical disease in humans. Our study is the first to demonstrate that human chondrocyte cells are permissive to RRV infection, support the production of infectious virus, and produce soluble factors including HPSE, which may contribute to joint degradation and the pathogenesis of disease.

Human Metapneumovirus Infection in Chronic Obstructive Pulmonary Disease: Impact of Glucocorticosteroids and Interferon.

Background: Human metapneumovirus (hMPV) infection is implicated in exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Research into the pathogenesis of infection is restricted to animal models, and information about hMPV replication and inflammatory and immune responses in human disease is limited. Methods: Human primary bronchial epithelial cells (PBECs) from healthy and asthmatic subjects and those with COPD were infected with hMPV, with or without glucocorticosteroid (GCS) exposure. Viral replication, inflammatory and immune responses, and apoptosis were analyzed. We also determined whether adjuvant interferon (IFN) can blunt hMPV infection in vitro and in a murine model. Results: hMPV infected human PBECs and viral replication was enhanced in cells from patients with COPD. The virus induced gene expression of IFN-stimulated gene 56 (ISG56) and IFN-ß, as well as IFN-γ-inducible protein 10 (IP-10) and regulated on activation, normal T cell expressed and secreted (RANTES), and more so in cells from patients with COPD. GCS exposure enhanced hMPV replication despite increased IFN expression. Augmented virus replication associated with GCS was mediated by reduced apoptosis via induction of antiapoptotic genes. Adjuvant IFN treatment suppressed hMPV replication in PBECs and reduced hMPV viral titers and inflammation in vivo. Conclusions: hMPV infects human PBECs, eliciting innate and inflammatory responses. Replication is enhanced by GCS and adjuvant IFN is an effective treatment, restricting virus replication and proinflammatory consequences of hMPV infections.

Role of envelope N-linked glycosylation in Ross River virus virulence and transmission.

With an expanding geographical range and no specific treatments, human arthritogenic alphaviral disease poses a significant problem worldwide. Previous in vitro work with Ross River virus (RRV) demonstrated that alphaviral N-linked glycosylation contributes to type I IFN (IFN-αß) induction in myeloid dendritic cells. This study further evaluated the role of alphaviral N-linked glycans in vivo, assessing the effect of glycosylation on pathogenesis in a mouse model of RRV-induced disease and on viral infection and dissemination in a common mosquito vector, Aedes vigilax. A viral mutant lacking the E1-141 glycosylation site was attenuated for virus-induced disease, with reduced myositis and higher levels of IFN-γ induction at peak disease contributing to improved viral clearance, suggesting that glycosylation of the E1 glycoprotein plays a major role in the pathogenesis of RRV. Interestingly, RRV lacking E2-200 glycan had significantly reduced replication in the mosquito vector A. vigilax, whereas loss of either of the E1 or E2-262 glycans had little effect on the competence of the mosquito vector. Overall, these results indicate that glycosylation of the E1 and E2 glycoproteins of RRV provides important determinants of viral virulence and immunopathology in the mammalian host and replication in the mosquito vector.

Interferon response factors 3 and 7 protect against Chikungunya virus hemorrhagic fever and shock.

Chikungunya virus (CHIKV) infections can produce severe disease and mortality. Here we show that CHIKV infection of adult mice deficient in interferon response factors 3 and 7 (IRF3/7(-/-)) is lethal. Mortality was associated with undetectable levels of alpha/beta interferon (IFN-α/ß) in serum, ∼50- and ∼10-fold increases in levels of IFN-γ and tumor necrosis factor (TNF), respectively, increased virus replication, edema, vasculitis, hemorrhage, fever followed by hypothermia, oliguria, thrombocytopenia, and raised hematocrits. These features are consistent with hemorrhagic shock and were also evident in infected IFN-α/ß receptor-deficient mice. In situ hybridization suggested CHIKV infection of endothelium, fibroblasts, skeletal muscle, mononuclear cells, chondrocytes, and keratinocytes in IRF3/7(-/-) mice; all but the latter two stained positive in wild-type mice. Vaccination protected IRF3/7(-/-) mice, suggesting that defective antibody responses were not responsible for mortality. IPS-1- and TRIF-dependent pathways were primarily responsible for IFN-α/ß induction, with IRF7 being upregulated >100-fold in infected wild-type mice. These studies suggest that inadequate IFN-α/ß responses following virus infection can be sufficient to induce hemorrhagic fever and shock, a finding with implications for understanding severe CHIKV disease and dengue hemorrhagic fever/dengue shock syndrome.

Severe seasonal influenza in ferrets correlates with reduced interferon and increased IL-6 induction.

Even though ferrets are one of the principal animal models for influenza pathogenesis, the lack of suitable immunological reagents has so far limited their use in host response studies. Using recently established real-time PCR assays for a panel of ferret cytokines, we analyzed the local ferret immune response to human influenza isolates of the H1N1 and H3N2 subtypes that varied in their virulence. We observed that the severity of clinical signs correlated with gross- and histopathological changes in the lungs and was subtype-independent. Strains causing a mild disease were associated with a strong and rapid innate response and upregulation of IL-8, while severe infections were characterized by a lesser induction of type I and II interferons and strong IL-6 upregulation. These findings suggest that more virulent strains may interfere more efficiently with the host response at early disease stages.

Canine distemper virus uses both the anterograde and the hematogenous pathway for neuroinvasion.

Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.