Antifungal Triazole Posaconazole Targets an Early Stage of the Parechovirus A3 Life Cycle.

Viruses in species Parechovirus A (Picornaviridae) are associated with a wide variety of clinical manifestations. Parechovirus A3 (PeV-A3) is known to cause sepsis-like illness, meningitis, and encephalitis in infants and young children. To date, no specific therapies are available to treat PeV-A3-infected children. We had previously identified two FDA-cleared antifungal drugs, itraconazole (ITC) and posaconazole (POS), with potent and specific antiviral activity against PeV-A3. Time-of-addition and synchronized infection assays revealed that POS targets an early stage of the PeV-A3 life cycle. POS exerts an antiviral effect, evidenced by a reduction in viral titer following the addition of POS to Vero-P cells before infection, coaddition of POS and PeV-A3 to Vero-P cells, incubation of POS and PeV-A3 prior to Vero-P infection, and at attachment. POS exerts less of an effect on virus entry. A PeV-A3 enzyme-linked immunosorbent assay inhibition experiment, using an anti-PeV-A3 monoclonal antibody, suggested that POS binds directly to the PeV-A3 capsid. POS-resistant PeV-A3 strains developed by serial passage in the presence of POS acquired substitutions in multiple regions of the genome, including the capsid. Reverse genetics confirmed substitutions in capsid proteins VP0, VP3, and VP1 and nonstructural proteins 2A and 3A. Single mutants VP0_K66R, VP0_A124T, VP3_N88S, VP1_Y224C, 2A_S78L, and 3A_T1I were 4-, 9-, 12-, 34-, 51-, and 119-fold more resistant to POS, respectively, than the susceptible prototype strain. Our studies demonstrate that POS may be a valuable tool in developing an antiviral therapy for PeV-A3.

Characterization of Pathogenesis and Inflammatory Responses to Experimental Parechovirus Encephalitis.

Human parechovirus type 3 (PeV-A3) infection has been recognized as an emerging etiologic factor causing severe nerve disease or sepsis in infants and young children. But the neuropathogenic mechanisms of PeV-A3 remain unknown. To understand the pathogenesis of PeV-A3 infection in the neuronal system, PeV-A3-mediated cytopathic effects were analyzed in human glioblastoma cells and neuroblastoma cells. PeV-A3 induced interferons and inflammatory cytokine expression in these neuronal cells. The pronounced cytopathic effects accompanied with activation of death signaling pathways of apoptosis, autophagy, and pyroptosis were detected. A new experimental disease model of parechovirus encephalitis was established. In the disease model, intracranial inoculation with PeV-A3 in C57BL/6 neonatal mice showed body weight loss, hindlimb paralysis, and approximately 20% mortality. PeV-A3 infection in the hippocampus and cortex regions of the neonatal mouse brain was revealed. Mechanistic assay supported the in vitro results, indicating detection of PeV-A3 replication, inflammatory cytokine expression, and death signaling transduction in mouse brain tissues. These in vitro and in vivo studies revealed that the activation of death signaling and inflammation responses is involved in PeV-A3-mediated neurological disorders. The present results might account for some of the PeV-A3-associated clinical manifestations.

Antiviral effects of selected nucleoside analogues against human parechoviruses A1 and A3.

Parechoviruses A (HPeV, Picornaviridae) are neglected human pathogens that cause sepsis-like illness and severe neurological complications in infants. There are no antivirals available for the treatment of HPeV infections. We here report on cell-based assays that allow for medium-throughput antiviral screening of compound libraries against HPeV. The nucleoside viral polymerase inhibitor 2'-C-methylcytidine was identified as being an in vitro replication inhibitor of HPeV1 and HPeV3 that can serve as a reference molecule for further antiviral studies.

Antifungal azoles itraconazole and posaconazole exhibit potent in vitro antiviral activity against clinical isolates of parechovirus A3 (Picornaviridae).

Parechovirus A3 (Par-A3, formerly human parechovirus 3) is an emerging viral infection of the central nervous system in children. We used an automated, homogeneous, cell based assay to identify itraconazole and posaconazole as inhibitors of Par-A3, with antiviral activity below concentrations clinically attainable in pediatric patients. Currently, there is no approved antiviral treatment for Par-A3 infection, despite numerous reports of serious Par-A3 disease in neonates and infants.

Enteroviruses and Parechoviruses.

Infections with enteroviruses and human parechoviruses are highly prevalent, particularly in neonates, where they may cause substantial morbidity and mortality. Individuals with B-cell-related immunodeficiencies are at risk for severe enteroviral infections, usually a chronic and fatal meningoencephalitis. In transplant recipients and patients with malignancy, enterovirus infections typically involve the respiratory tract, but cases of severe, disseminated infection have been described. The mainstay of diagnosis for enterovirus and human parechovirus infections involves the use of molecular diagnostic techniques. However, routine nucleic acid-detection methods for enteroviruses will not detect human parechoviruses. Laboratory diagnosis of these viral infections is important in determining a patient's prognosis and guiding clinical management.

Replication and Inhibitors of Enteroviruses and Parechoviruses.

The Enterovirus (EV) and Parechovirus genera of the picornavirus family include many important human pathogens, including poliovirus, rhinovirus, EV-A71, EV-D68, and human parechoviruses (HPeV). They cause a wide variety of diseases, ranging from a simple common cold to life-threatening diseases such as encephalitis and myocarditis. At the moment, no antiviral therapy is available against these viruses and it is not feasible to develop vaccines against all EVs and HPeVs due to the great number of serotypes. Therefore, a lot of effort is being invested in the development of antiviral drugs. Both viral proteins and host proteins essential for virus replication can be used as targets for virus inhibitors. As such, a good understanding of the complex process of virus replication is pivotal in the design of antiviral strategies goes hand in hand with a good understanding of the complex process of virus replication. In this review, we will give an overview of the current state of knowledge of EV and HPeV replication and how this can be inhibited by small-molecule inhibitors.

Genome and infection characteristics of human parechovirus type 1: the interplay between viral infection and type I interferon antiviral system.

Human parechoviruses (HPeVs), members of the family Picornaviridae, are associated with severe human clinical conditions such as gastrointestinal disease, encephalitis, meningitis, respiratory disease and neonatal sepsis. A new contemporary strain of HPeV1, KVP6 (accession no. KC769584), was isolated from a clinical specimen. Full-genome alignment revealed that HPeV1 KVP6 shares high genome homology with the German strain of HPeV1, 7555312 (accession no. FM178558) and could be classified in the clade 1B group. An intertypic recombination was shown within the P2-P3 genome regions of HPeV1. Cell-type tropism test showed that T84 cells (colon carcinoma cells), A549 cells (lung carcinoma cells) and DBTRG-5MG cells (glioblastoma cells) were susceptible to HPeV1 infection, which might be relevant clinically. A facilitated cytopathic effect and increased viral titers were reached after serial viral passages in Vero cells, with viral genome mutation found in later passages. HPeV1 is sensitive to elevated temperature because 39C incubation impaired virion production. HPeV1 induced innate immunity with phosphorylation of interferon (IFN) regulatory transcription factor 3 and production of type I IFN in A549 but not T84 cells. Furthermore, type I IFN inhibited HPeV1 production in A549 cells but not T84 cells; T84 cells may be less responsive to type I IFN stimulation. Moreover, HPeV1-infected cells showed downregulated type I IFN activation, which indicated a type I IFN evasion mechanism. The characterization of the complete genome and infection features of HPeV1 provide comprehensive information about this newly isolated HPeV1 for further diagnosis, prevention or treatment strategies.

Pleconaril-resistant chronic parechovirus-associated enteropathy in agammaglobulinaemia.

A 14 year old common variable immunodeficiency patient developed severe protein-losing enteropathy. A chronic enteral infection with human parechovirus type 1 and norovirus was diagnosed. Treatment strategies aimed at virus eradication and providing supportive care were ineffective. The antipicornavirus agent pleconaril did not have any effect on viral replication. Symptoms improved on immunosuppressive therapy, suggesting infection-related immune dysregulation in an immunocompromised host.

A model system for in vitro studies of bank vole borne viruses.

The bank vole (Myodes glareolus) is a common small mammal in Europe and a natural host for several important emerging zoonotic viruses, e.g. Puumala hantavirus (PUUV) that causes hemorrhagic fever with renal syndrome (HFRS). Hantaviruses are known to interfere with several signaling pathways in infected human cells, and HFRS is considered an immune-mediated disease. There is no in vitro-model available for infectious experiments in bank vole cells, nor tools for analyses of bank vole immune activation and responses. Consequently, it is not known if there are any differences in the regulation of virus induced responses in humans compared to natural hosts during infection. We here present an in vitro-model for studies of bank vole borne viruses and their interactions with natural host cell innate immune responses. Bank vole embryonic fibroblasts (VEFs) were isolated and shown to be susceptible for PUUV-infection, including a wild-type PUUV strain (only passaged in bank voles). The significance of VEFs as a model system for bank vole associated viruses was further established by infection studies showing that these cells are also susceptible to tick borne encephalitis, cowpox and Ljungan virus. The genes encoding bank vole IFN-ß and Mx2 were partially sequenced and protocols for semi-quantitative RT-PCR were developed. Interestingly, PUUV did not induce an increased IFN-ß or Mx2 mRNA expression. Corresponding infections with CPXV and LV induced IFN-ß but not Mx2, while TBEV induced both IFN-ß and Mx2. In conclusion, VEFs together with protocols developed for detection of bank vole innate immune activation provide valuable tools for future studies of how PUUV and other zoonotic viruses affect cells derived from bank voles compared to human cells. Notably, wild-type PUUV which has been difficult to cultivate in vitro readily infected VEFs, suggesting that embryonic fibroblasts from natural hosts might be valuable for isolation of wild-type hantaviruses.

The need for treatment against human parechoviruses: how, why and when?

Since 1999, human parechoviruses (HPeVs) have been classified as a separate group in the large and expanding family of Picornaviridae. In contrast to the well-established group of the human enteroviruses (HEVs), HPeVs have long been considered as irrelevant and have only been associated with mild disease manifestations in children. The identification of HPeV-3 in 2004 and its association with neonatal sepsis, refocused attention on this neglected group of viruses. Clinically HPeV infections may mimic HEV infections and are increasingly recognized as viral causes of sepsis-like illness and CNS infections in young children. Therapy is not available against HPeVs or HEVs. In this article, we will demonstrate that therapy against this group of picornaviruses is urgently needed and we will review the current knowledge of treatment options as well as the current developments in antiviral therapy against picornaviruses in the scope of treatment possibilities against HPeVs.

Antiviral treatments reduce severity of diabetes in Ljungan virus-infected CD-1 mice and delay onset in diabetes-prone BB rats.

The effects of LV in two different species, CD-1 mice, without a genetic disposition for diabetes, and BB rats prone to T1D were examined. Male CD-1 mice that had been exposed to LV in utero developed a type 2-like diabetes with increased blood glucose, insulin levels and epididymal fat at the age of 10-15 weeks. Combination therapy including LV-antiserum and an antiviral drug, Pleconaril, significantly reduced the levels of blood glucose and insulin and the amount of abdominal fat. In BB rats, LV has been found in both prediabetic- and diabetic diabetes-prone rats, as well as in diabetes-resistant rats. To evaluate whether the presence of LV has any influence on the onset of T1D, prediabetic BB rats were treated with an antiserum against LV or a combination of the antiviral drugs Pleconaril and Ribavirin. In the group treated with antiviral drugs, the onset was significantly delayed. These results indicate that the presence of LV can be involved in the pathogenesis of diabetes in these animal models.

Differential requirements for COPI coats in formation of replication complexes among three genera of Picornaviridae.

Picornavirus RNA replication requires the formation of replication complexes (RCs) consisting of virus-induced vesicles associated with viral nonstructural proteins and RNA. Brefeldin A (BFA) has been shown to strongly inhibit RNA replication of poliovirus but not of encephalomyocarditis virus (EMCV). Here, we demonstrate that the replication of parechovirus 1 (ParV1) is partly resistant to BFA, whereas echovirus 11 (EV11) replication is strongly inhibited. Since BFA inhibits COPI-dependent steps in endoplasmic reticulum (ER)-Golgi transport, we tested a hypothesis that different picornaviruses may have differential requirements for COPI in the formation of their RCs. Using immunofluorescence and cryo-immunoelectron microscopy we examined the association of a COPI component, beta-COP, with the RCs of EMCV, ParV1, and EV11. EMCV RCs did not contain beta-COP. In contrast, beta-COP appeared to be specifically distributed to the RCs of EV11. In ParV1-infected cells beta-COP was largely dispersed throughout the cytoplasm, with some being present in the RCs. These results suggest that there are differences in the involvement of COPI in the formation of the RCs of various picornaviruses, corresponding to their differential sensitivity to BFA. EMCV RCs are likely to be formed immediately after vesicle budding from the ER, prior to COPI association with membranes. ParV1 RCs are formed from COPI-containing membranes but COPI is unlikely to be directly involved in their formation, whereas formation of EV11 RCs appears to be dependent on COPI association with membranes.