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Objective Saffold virus (SAFV), a picornavirus, is occasionally detected in children with acute flaccid paralysis, meningitis, and cerebellitis; however, the neuropathogenicity of SAFV remains undetermined. Methods The virulence of two clinical isolates of SAFV type 3 (SAFV-3) obtained from a patient with aseptic meningitis (AM strain) and acute upper respiratory inflammation (UR strain) was analyzed in neonatal and young mice utilizing virological, pathological, and immunological methods. Results The polyproteins of the strains differed in eight amino acids. Both clinical isolates were infective, exhibited neurotropism, and were mildly neurovirulent in neonatal ddY mice. Both strains pathologically infected neural progenitor cells and glial cells, but not large neurons, with the UR strain also infecting epithelial cells. UR infection resulted in longer inflammation in the brain and spinal cord because of demyelination, while the AM strain showed more infectivity in the cerebellum in neonatal ddY mice. Additionally, young BALB/c mice seroconverted following mucosal inoculation with the UR, but not the AM, strain. Conclusions Both SAFV-3 isolates had neurotropism and mild neurovirulence but showed different cell tropisms in both neonatal and young mouse models. This animal model has the potential to recapitulate the potential neuropathogenicity of SAFV-3.

Unlike traditional virus isolation and sequencing approaches, sequence-independent amplification based viral metagenomics technique allows one to discover unexpected or novel viruses efficiently while bypassing culturing step. Here we report the discovery of the first Sicinivirus isolate (designated as strain JSY) of picornaviruses from commercial layer chickens in mainland China by using a viral metagenomics technique. This Sicinivirus isolate, which contains a whole genome of 9,797 nucleotides (nt) excluding the poly(A) tail, possesses one of the largest picornavirus genome so far reported, but only shares 88.83% and 82.78% of amino acid sequence identity to that of ChPV1 100C (KF979332) and Sicinivirus 1 strain UCC001 (NC_023861), respectively. The complete 939 nt 5'UTR of the isolate strain contains at least twelve stem-loop domains (A-L), representing the highest set of loops reported within Sicinivirus genus. The conserved 'barbell-like' structure was also present in the 272 nt 3'UTR of the isolate as that in the 3' UTR of Sicinivirus 1 strain UCC001. The 8,586 nt large open reading frame encodes a 2,862 amino acids polyprotein precursor. Moreover, Sicinivirus infection might be widely present in commercial chicken farms in Yancheng region of the Jiangsu Province as evidenced by all the tested stool samples from three different farms being positive (17/17) for Sicinivirus detection. This is the first report on identification of Sicinivirus in commercial layer chickens with a severe clinical disease in mainland China, however, further studies are needed to evaluate the pathogenic potential of this picornavirus in chickens.

The oncolytic picornavirus Seneca Valley Virus (SVV-001) demonstrates anti-tumor activity in models of small cell lung cancer (SCLC), but may ultimately need to be combined with cytotoxic therapies to improve responses observed in patients. Combining SVV-001 virotherapy with a peptide prodrug activated by the viral protease 3Cpro is a novel strategy that may increase the therapeutic potential of SVV-001. Using recombinant SVV-001 3Cpro, we measured cleavage kinetics of predicted SVV-001 3Cpro substrates. An efficient substrate, L/VP4 (kcat/KM = 1932 plus or minus 183 M-1s-1), was further optimized by a P2' N arrow right P substitution yielding L/VP4.1 (kcat/KM = 17446 plus or minus 2203 M-1s-1). We also determined essential substrate amino acids by sequential N-terminal deletion and substitution of amino acids found in other picornavirus genera. A peptide corresponding to the L/VP4.1 substrate was selectively cleaved by SVV-001 3Cpro in vitro and was stable in human plasma. These data define an optimized peptide substrate for SVV-001 3Cpro, with direct implications for anti-cancer therapeutic development.

Glioblastoma (GBM) is among the most invasive and lethal of cancers, frequently infiltrating surrounding healthy tissue and giving rise to rapid recurrence. It is therefore critical to establish experimental model systems and develop therapeutic approaches that enhance anti-tumor immunity. In the current study, we have employed a newly developed murine glioma model to assess the efficacy of a novel picornavirus vaccination approach for the treatment of established tumors. The GL261-Quad system is a variation of the GL261 syngeneic glioma that has been engineered to expresses model T cell epitopes including OVA257-264. MRI revealed that both GL261 and GL261-Quad tumors display characteristic features of human gliomas such as heterogeneous gadolinium leakage and larger T2 weighted volumes. Analysis of brain-infiltrating immune cells demonstrated that GL261-Quad gliomas generate detectable CD8+ T cell responses toward the tumor-specific Kb:OVA257-264 antigen. Enhancing this response via a single intracranial or peripheral vaccination with picornavirus expressing the OVA257-264 antigen increased anti-tumor CD8+ T cells infiltrating the brain, attenuated progression of established tumors, and extended survival of treated mice. Importantly, the efficacy of the picornavirus vaccination is dependent on functional cytotoxic activity of CD8+ T cells, as the beneficial response was completely abrogated in mice lacking perforin expression. Therefore, we have developed a novel system for evaluating mechanisms of anti-tumor immunity in vivo, incorporating the GL261-Quad model, 3D volumetric MRI, and picornavirus vaccination to enhance tumor-specific cytotoxic CD8+ T cell responses and track their effectiveness at eradicating established gliomas in vivo.

Porcine circovirus 2 (PCV-2) is one of the most widespread viral infections of swine, causing a remarkable economic impact because of direct losses and indirect costs for its control. As other ssDNA viruses, PCV-2 is characterized by a high evolutionary rate, leading to the emergence of a plethora of variants with different biological and epidemiological features. Over time, several attempts have been made to organize PCV-2 genetic heterogeneity in recognized genotypes. This categorization has clearly simplified the epidemiological investigations, allowing to identify different spatial and temporal patterns among genotypes. Additionally, variable virulence and vaccine effectiveness have also been hypothesized. However, the rapid increase in sequencing activity, coupled with the per se high viral variability, has challenged the previously established nomenclature, leading to the definition of several study-specific genotypes and hindering the capability of performing comparable epidemiological studies. Based on these premises, an updated classification scheme is herein reported. Recognizing the impossibility of defining a clear inter-cluster p-distance cut-off, the present study proposes a phylogeny-grounded genotype definition based on three criteria: maximum intra-genotype p-distance of 13% (calculated on the ORF2 gene), bootstrap support at the corresponding internal node higher than 70% and at least 15 available sequences. This scheme allowed defining 8 genotypes (PCV-2a to PCV-2h), which six of those had been previously proposed. To minimize the inconvenience of implementing a new classification, the most common names already adopted have been maintained when possible. The analysis of sequence-associated metadata highlighted a highly unbalanced sequencing activity in terms of geographical, host and temporal distribution. The PCV-2 molecular epidemiology scenario appears therefore characterized by a severe bias that could lead to spurious associations between genetic and epidemiological/biological viral features. While the suggested classification can establish a \"common language\" for future studies, further efforts should be paid to achieve a more homogeneous and informative representation of the PCV-2 global scenario.

A wide range of viruses from different virus families in different geographical areas, may cause immediate or delayed neuropathological changes and neurological manifestations in humans and animals. Infection by neurotropic viruses as well as the resulting immune response can irreversibly disrupt the complex structural and functional architecture of the central nervous system, frequently leaving the patient or affected animal with a poor or fatal prognosis. Mechanisms that govern neuropathogenesis and immunopathogenesis of viral infections are highlighted, using examples of well-studied virus infections that are associated with these alterations in different populations throughout the world. A better understanding of the molecular, epidemiological and biological characteristics of these infections and in particular of mechanisms that underlie their clinical manifestations may be expected to provide tools for the development of more effective intervention strategies and treatment regimens.

IntroductionNKT can enhance anti-pathogen and anti-tumour immunity via NK and DC, leading to T/B cell activation. Th1-biassed NKT can contribute to protective responses against cancer and viruses. However, NKT can contribute to immunopathology in chronic infections. Patient NKT defects are reversible in vitro, so we are investigating correcting such defects in models and clinic.NKT are required for optimal resistance to certain viruses. Mice lacking all NKT are impaired in lymphocyte activation and in picornavirus clearance. Conversely to acute infection, chronic viral stimulation of NKT can lead to immunopathology. CD1d-specific gamma delta T cells respond to CD1d induced by infection, causing viral myocarditis.MethodsModel liver and human hepatic immune responses were measured. CD1d reactivity of distinct T cell populations was assayed, compared to total T cell responses.ResultsNKT also potentially contribute to chronic Hepatitis C, since human liver-specific gamma delta V delta 3 T cells were CD1d-reactive. CD1d is up-regulated by HCV and high level hepatic CD1d-reactive NKT produce pro-inflammatory Th1 cytokines in chronic hepatitis C and their Type 2 responses increase in cirrhosis.ConclusionNKT in Chronic Hepatitis C, instead of being protective, may promote inflammation and fibrosis while suppressing anti-tumour responses: their inhibition might have clinical utility.Disclosure of InterestM. Exley Shareholder of: NKT Therapeutics, Inc.

Cyclic GMP-AMP synthase (cGAS) plays a key role in the innate immune responses to both DNA and RNA virus infection. Here, we found that enterovirus 71 (EV-A71), Seneca Valley virus (SVV), and foot-and-mouth disease virus (FMDV) infection triggered mitochondria damage and mitochondrial DNA (mtDNA) release in vitro and vivo . These responses were mediated by picornavirus 2B proteins which induced mtDNA release during viral replication. SVV infection caused the opening of mitochondrial permeability transition pore (mPTP) and led to voltage-dependent anion channel 1 (VDAC1)- and BCL2 antagonist/killer 1 (Bak) and Bak/BCL2-associated X (Bax)-dependent mtDNA leakage into the cytoplasm, while EV-A71 and FMDV infection induced mPTP opening and resulted in VDAC1-dependent mtDNA release. The released mtDNA bound to cGAS and activated cGAS-mediated antiviral immune response. cGAS was essential for inhibiting EV-A71, SVV, and FMDV replication by regulation of IFN-β production. cGAS deficiency contributed to higher mortality of EV-A71- or FMDV-infected mice. In addition, we found that SVV 2C protein was responsible for decreasing cGAS expression through the autophagy pathway. The 9th and 153rd amino acid sites in 2C were critical for induction of cGAS degradation. Furthermore, we also show that EV-A71, CA16, and EMCV 2C antagonize the cGAS-stimulator of interferon genes (STING) pathway through interaction with STING, and highly conserved amino acids Y155 and S156 were critical for this inhibitory effect. In conclusion, these data reveal novel mechanisms of picornaviruses to block the antiviral effect mediated by the cGAS-STING signaling pathway, which will provide insights for developing antiviral strategies against picornaviruses.

The Picornaviridae family comprises a large group of non-enveloped viruses with enormous impact on human and animal health. The picornaviral genome contains one open reading frame encoding a single polyprotein that can be processed by viral proteases. The picornaviral 3C proteases share similar three-dimensional structures and play a significant role in the viral life cycle and virus–host interactions. Picornaviral 3C proteins also have conserved RNA-binding activities that contribute to the assembly of the viral RNA replication complex. The 3C protease is important for regulating the host cell response through the cleavage of critical host cell proteins, acting to selectively ‘hijack’ host factors involved in gene expression, promoting picornavirus replication, and inactivating key factors in innate immunity signaling pathways. The protease and RNA-binding activities of 3C are involved in viral polyprotein processing and the initiation of viral RNA synthesis. Most importantly, 3C modifies critical molecules in host organelles and maintains virus infection by subtly subverting host cell death through the blocking of transcription, translation, and nucleocytoplasmic trafficking to modulate cell physiology for viral replication. Here, we discuss the molecular mechanisms through which 3C mediates physiological processes involved in promoting virus infection, replication, and release.

The picornavirus 3CD protein is a precursor to the 3C main protease and the 3D RNA-dependent RNA polymerase. In addition to its functions in proteolytic processing of the virus polyprotein and cleavage of key host factors, the 3C domain interacts with cis-acting replication elements (CREs) within the viral genome to regulate replication and translation events. We investigated the molecular determinants of RNA binding to 3C using a wide range of biophysical and computational methods. These studies showed that 3C binds to a broad spectrum of RNA oligonucleotides, displaying minimal sequence and structure dependence, at least for these shorter RNAs. However, they also uncovered a novel aspect of these interactions, that is, 3C-RNA binding can induce liquid–liquid phase separation (LLPS), with 3CD–RNA interactions likewise leading to LLPS. This may be a general phenomenon for other 3C and 3C-like proteases and polyproteins incorporating 3C domains. These findings have potential implications in understanding virally induced apoptosis and the control of stress granules, which involve LLPS and include other proteins with known interactions with 3C/3CD.