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Human norovirus (HuNV) is a leading cause of acute gastroenteritis worldwide with most infections caused by genogroup I and genogroup II (GII) viruses. Replication of HuNV generates both precursor and mature proteins during processing of the viral polyprotein that are essential to the viral lifecycle. One such precursor is protease-polymerase (ProPol), a multi-functional enzyme comprised of the norovirus protease and polymerase proteins. This work investigated HuNV ProPol by determining the de novo polymerase activity, protein structure, and antiviral inhibition profile. The GII ProPol de novo enzymatic efficiencies ( k cat / K m ) for RNA templates and ribonucleotides were equal or superior to those of mature GII Pol on all templates measured. Furthermore, GII ProPol was the only enzyme form active on a poly(A) template. The first structure of the polymerase domain of HuNV ProPol in the unliganded state was determined by cryo-electron microscopy at a resolution of 2.6 Å. The active site and overall architecture of ProPol are similar to those of mature Pol. In addition, both galidesivir triphosphate and PPNDS inhibited polymerase activity of GII ProPol, with respective half-maximal inhibitory concentration (IC 50 ) values of 247.5 µM and 3.8 µM. In both instances, the IC 50 obtained with ProPol was greater than that of mature Pol, indicating that ProPol can exhibit different responses to antivirals. This study provides evidence that HuNV ProPol possesses overlapping and unique enzyme properties compared with mature Pol and will aid our understanding of the replication cycle of the virus.IMPORTANCEDespite human norovirus (HuNV) being a leading cause of acute gastroenteritis, the molecular mechanisms surrounding replication are not well understood. Reports have shown that HuNV replication generates precursor proteins from the viral polyprotein, one of which is the protease-polymerase (ProPol). This precursor is important for viral replication; however, the polymerase activity and structural differences between the precursor and mature forms of the polymerase remain to be determined. We show that substrate specificity and polymerase activity of ProPol overlap with, but is distinct from, the mature polymerase. We employ cryo-electron microscopy to resolve the first structure of the polymerase domain of ProPol. This shows a polymerase architecture similar to mature Pol, indicating that the interaction of the precursor with substrates likely defines its activity. We also show that ProPol responds differently to antivirals than mature polymerase. Altogether, these findings enhance our understanding of the function of the important norovirus ProPol precursor., Competing Interests: The authors declare no conflict of interest.

An intrinsically disordered protein (IDP) or region (IDR) lacks or has little protein structure but still maintains function. This lack of structure creates flexibility and fluidity, allowing multiple protein conformations and potentially transient interactions with more than one partner. Caliciviruses are positive-sense ssRNA viruses, containing a relatively small genome of 7.6-8.6 kb and have a broad host range. Many viral proteins are known to contain IDRs, which benefit smaller viral genomes by expanding the functional proteome through the multifunctional nature of the IDR. The percentage of intrinsically disordered residues within the total proteome for each calicivirus type species can range between 8 and 23%, and IDRs have been experimentally identified in NS1-2, VPg and RdRP proteins. The IDRs within a protein are not well conserved across the genera, and whether this correlates to different activities or increased tolerance to mutations, driving virus adaptation to new selection pressures, is unknown. The function of norovirus NS1-2 has not yet been fully elucidated but includes involvement in host cell tropism, the promotion of viral spread and the suppression of host interferon-λ responses. These functions and the presence of host cell-like linear motifs that interact with host cell caspases and VAPA/B are all found or affected by the disordered region of norovirus NS1-2. The IDRs of calicivirus VPg are involved in viral transcription and translation, RNA binding, nucleotidylylation and cell cycle arrest, and the N-terminal IDR within the human norovirus RdRP could potentially drive liquid-liquid phase separation. This review identifies and summarises the IDRs of proteins within the Caliciviridae family and their importance during viral replication and subsequent host interactions.

Norovirus is the leading cause of viral gastroenteritis worldwide, and there are no approved vaccines or therapeutic treatments for chronic or severe norovirus infections. The structural characterisation of the norovirus protease and drug development has predominantly focused upon GI.1 noroviruses, despite most global outbreaks being caused by GII.4 noroviruses. Here, we determined the crystal structures of the GII.4 Sydney 2012 ligand-free norovirus protease at 2.79 Å and at 1.83 Å with a covalently bound high-affinity (IC 50 = 0.37 µM) protease inhibitor (NV-004). We show that the active sites of the ligand-free protease structure are present in both open and closed conformations, as determined by their Arg112 side chain orientation. A comparative analysis of the ligand-free and ligand-bound protease structures reveals significant structural differences in the active site cleft and substrate-binding pockets when an inhibitor is covalently bound. We also report a second molecule of NV-004 non-covalently bound within the S4 substrate binding pocket via hydrophobic contacts and a water-mediated hydrogen bond. These new insights can guide structure-aided drug design against the GII.4 genogroup of noroviruses.

Murine norovirus (MNV) is a positive-sense, plus-stranded RNA virus in the Caliciviridae family. Viruses in this family replicate in the intestine and are transmitted by the fecal-oral route. MNV is related to the human noroviruses, which cause the majority of nonbacterial gastroenteritis worldwide. Given the technical challenges in studying human norovirus, MNV is often used to study mechanisms in norovirus biology since it combines the availability of a cell culture and reverse genetics system with the ability to study infection in the native host. Adding to our previous protocol collection, here we describe additional techniques that have since been developed to study MNV biology. © 2023 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Indirect method for measuring cell cytotoxicity and antiviral activity Basic Protocol 2: Measuring murine norovirus genome titers by RT-qPCR Support Protocol 1: Preparation of standard Basic Protocol 3: Generation of recombinant murine norovirus with minimal passaging Basic Protocol 4: Generation of recombinant murine norovirus via circular polymerase extension reaction (CPER) Basic Protocol 5: Expression of norovirus NS1-2 in insect cell suspension cultures using a recombinant baculovirus Support Protocol 2: Isotope labelling of norovirus NS1-2 in insect cells Support Protocol 3: Purification of the norovirus NS1-2 protein Support Protocol 4: Expression of norovirus NS1-2 in mammalian cells by transduction with a recombinant baculovirus Basic Protocol 6: Infection of enteroids in transwell inserts with murine norovirus Support Protocol 5: Preparation of conditioned medium for enteroids culture Support Protocol 6: Isolation of crypts for enteroids generation Support Protocol 7: Enteroid culture passaging and maintenance Basic Protocol 7: Quantification of murine norovirus-induced diarrhea using neonatal mouse infections Alternate Protocol 1: Intragastric inoculation of neonatal mice Alternate Protocol 2: Scoring colon contents., (© 2023 The Authors. Current Protocols published by Wiley Periodicals LLC.)

Viral infections are one of the leading causes of acute morbidity in humans and much endeavour has been made by the synthetic community for the development of drugs to treat associated diseases. Peptide-based enzyme inhibitors, usually short sequences of three or four residues, are one of the classes of compounds currently under development for enhancement of their activity and pharmaceutical properties. This review reports the advances made in the design of inhibitors targeting the family of highly conserved viral proteases 3C/3CL pro , which play a key role in viral replication and present minimal homology with mammalian proteases. Particular focus is put on the reported development of P 1 glutamine isosteres to generate potent inhibitors mimicking the natural substrate sequence at the site of recognition.', Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

We report for the first time the antiviral activities of two iminovirs (antiviral imino- C -nucleosides) 1 and 2 , structurally related to galidesivir (Immucillin A, BCX4430). An iminovir containing the 4-aminopyrrolo[2,1- f ][1,2,4-triazine] nucleobase found in remdesivir exhibited submicromolar inhibition of multiple strains of influenza A and B viruses, as well as members of the Bunyavirales order. We also report the first syntheses of ProTide prodrugs of iminovir monophosphates, which unexpectedly displayed poorer viral inhibition than their parent nucleosides in vitro . An efficient synthesis of the 4-aminopyrrolo[2,1- f ][1,2,4-triazine]-containing iminovir 2 was developed to enable preliminary in vivo studies, wherein it displayed significant toxicity in BALB/c mice and limited protection against influenza. Further modification of this anti-influenza iminovir will therefore be required to improve its therapeutic value., Competing Interests: The authors declare no competing financial interest., (© 2023 American Chemical Society.)

Akt (protein kinase B) is a key signaling protein in eukaryotic cells that controls many cellular processes, such as glucose metabolism and cell proliferation, for survival. As obligate intracellular pathogens, viruses modulate host cellular processes, including Akt signaling, for optimal replication. The mechanisms by which viruses modulate Akt and the resulting effects on the infectious cycle differ widely depending on the virus. In this study, we explored the effect of Akt serine 473 phosphorylation (p-Akt) during murine norovirus (MNV) infection. p-Akt increased during infection of murine macrophages with acute MNV-1 and persistent CR3 and CR6 strains. Inhibition of Akt with MK2206, an inhibitor of all three isoforms of Akt (Akt1/2/3), reduced infectious virus progeny of all three virus strains. This reduction was due to decreased viral genome replication (CR3), defective virus assembly (MNV-1), or altered cellular egress (CR3 and CR6) in a virus strain-dependent manner. Collectively, our data demonstrate that Akt activation increases in macrophages during the later stages of the MNV infectious cycle, which may enhance viral infection in unique ways for different virus strains. The data, for the first time, indicate a role for Akt signaling in viral assembly and highlight additional phenotypic differences between closely related MNV strains. IMPORTANCE Human noroviruses (HNoV) are a leading cause of viral gastroenteritis, resulting in high annual economic burden and morbidity, yet there are no small-animal models supporting productive HNoV infection or robust culture systems producing cell culture-derived virus stocks. As a result, research on drug discovery and vaccine development against norovirus infection has been challenging, and no targeted antivirals or vaccines against HNoV are approved. On the other hand, murine norovirus (MNV) replicates to high titers in cell culture and is a convenient and widespread model in norovirus research. Our data demonstrate the importance of Akt signaling during the late stage of the MNV life cycle. Notably, the effect of Akt signaling on genome replication, virus assembly, and cellular egress is virus strain specific, highlighting the diversity of biological phenotypes despite small genetic variability among norovirus strains. This study is the first to demonstrate a role for Akt in viral assembly.

Nucleotidylylation is a post-transcriptional modification important for replication in the picornavirus supergroup of RNA viruses, including members of the Caliciviridae , Coronaviridae , Picornaviridae and Potyviridae virus families. This modification occurs when the RNA-dependent RNA polymerase (RdRp) attaches one or more nucleotides to a target protein through a nucleotidyl-transferase reaction. The most characterized nucleotidylylation target is VPg (viral protein genome-linked), a protein linked to the 5' end of the genome in Caliciviridae , Picornaviridae and Potyviridae . The nucleotidylylation of VPg by RdRp is a critical step for the VPg protein to act as a primer for genome replication and, in Caliciviridae and Potyviridae, for the initiation of translation. In contrast, Coronaviridae do not express a VPg protein, but the nucleotidylylation of proteins involved in replication initiation is critical for genome replication. Furthermore, the RdRp proteins of the viruses that perform nucleotidylylation are themselves nucleotidylylated, and in the case of coronavirus, this has been shown to be essential for viral replication. This review focuses on nucleotidylylation within the picornavirus supergroup of viruses, including the proteins that are modified, what is known about the nucleotidylylation process and the roles that these modifications have in the viral life cycle.

The viral protein genome-linked (VPg) of noroviruses is a multi-functional protein that participates in essential roles during the viral replication cycle. Predictive analyses indicate that murine norovirus (MNV) VPg contains a disordered N-terminal region with RNA binding potential. VPg proteins were expressed with an N-terminal spidroin fusion protein in insect cells and the interaction with RNA investigated by electrophoretic mobility shift assays (EMSA) against a series of RNA probes (pentaprobes) representing all possible five nucleotide combinations. MNV VPg and human norovirus (HuNV) VPg proteins were directly bound to RNA in a non-specific manner. To identify amino acids involved in binding to RNA, all basic (K/R) residues in the first 12 amino acids of MNV VPg were mutated to alanine. Removal of the K/R amino acids eliminated RNA binding and is consistent with a K/R basic patch RNA binding motif within the disordered N-terminal region of norovirus VPgs. Finally, we show that mutation of the K/R basic patch required for RNA binding eliminates the ability of MNV VPg to induce a G0/G1 cell cycle arrest.

Breast cancer (BC) is the most frequently diagnosed cancer in women, with many patients experiencing recurrence following treatment. Antigens delivered on virus-like particles (VLPs) induce a targeted immune response and here we investigated whether the co-delivery of multiple antigens could induce a superior anti-cancer response for BC immunotherapy. VLPs were designed to recombinantly express murine survivin and conjugated with an aberrantly glycosylated mucin-1 (MUC1) peptide using an intracellular cleavable bis-arylhydrazone linker. Western blotting, electron microscopy and UV absorption confirmed survivin-VLP expression and MUC1 conjugation. To assess the therapeutic efficacy of VLPs, orthotopic BC tumours were established by injecting C57mg.MUC1 cells into the mammary fat pad of mice, which were then vaccinated with surv.VLP-SS-MUC1 or VLP controls. While wild-type mice vaccinated with surv.VLP-SS-MUC1 showed enhanced survival compared to VLPs delivering either antigen alone, MUC1 transgenic mice vaccinated with surv.VLP-SS-MUC1 showed no enhanced survival compared to controls. Hence, while co-delivery of two tumour antigens on VLPs can induce a superior anti-tumour immune response compared to the delivery of single antigens, additional strategies must be employed to break tolerance when targeted tumour antigens are expressed as endogenous self-proteins. Using VLPs for the delivery of multiple antigens represents a promising approach to improving BC immunotherapy, and has the potential to be an integral part of combination therapy in the future.

Biologics can be combined with liquid polymer materials and electrospun to produce a dry nanofibrous scaffold. Unlike spray-drying and freeze-drying, electrospinning minimizes the physiological stress on sensitive materials, and nanofiber mat properties such as hydrophobicity, solubility, and melting temperature can be tuned based on the polymer composition. In this study, we explored the dry formulation of a virus-like particle (VLP) vaccine by electrospinning VLP derived from rabbit hemorrhagic disease virus modified to carry the MHC-I gp100 tumor-associated antigen epitope. VLP were added to a polyvinylpyrrolidone (PVP) solution (15% w / v ) followed by electrospinning at 24 kV. Formation of a nanofibrous mat was confirmed by scanning electron microscopy, and the presence of VLP was confirmed by transmission electron microscopy and Western blot. VLP from the nanofibers induced T-cell activation and interferon- (IFN-) γ production in vitro. To confirm in vivo cytotoxicity, Pmel mice treated by injection with gp100 VLP from nanofibers induced a gp100 specific immune response, lysing approximately 65% of gp100-pulsed target cells, comparable to mice vaccinated with gp100 VLP in PBS. VLP from nanofibers also induced an antibody response. This work shows that electrospinning can be used to dry-formulate VLP, preserving both humoral and cell-mediated immunity.

Felis catus papillomavirus type 2 (FcaPV-2) commonly infects the skin of domestic cats and has been associated with the development of skin cancer. In the present study, a FcaPV-2 virus-like particle (VLP) vaccine was produced and assessed for vaccine safety, immunogenicity, and impact on FcaPV-2 viral load. This is the first report of the use of a papillomavirus VLP vaccine in domestic cats. The FcaPV-2 VLP vaccine was given to ten adult cats that were naturally infected with FcaPV-2, and a further ten naturally infected cats were sham vaccinated as a control group. The rationale for vaccinating cats already infected with the virus was to induce neutralizing antibody titers that could prevent reinfection of new areas of skin and reduce the overall viral load, as has been demonstrated in other species. Reducing the overall FcaPV-2 viral load could reduce the risk for subsequent PV-associated cancer. The vaccine in this study was well-tolerated, as none of the cats developed any signs of local reaction or systemic illness. In the treatment group, the geometric mean anti-papillomavirus endpoint antibody titers increased significantly following vaccination from 606 (95% CI 192-1913) to 4223 (2023-8814), a 7.0-fold increase, although the individual antibody response varied depending on the level of pre-existing antibodies. Despite the immunogenicity of the vaccine, there was no significant change in FcaPV-2 viral load in the treatment group compared to the control group, over the 24 week follow-up period. A possible reason is that FcaPV-2 was already widespread in the basal skin layer of these adult cats and so preventing further cells from becoming infected had no impact on the overall viral load. Therefore, these results do not support the use of a FcaPV-2 VLP vaccine to reduce the risk for PV-associated cancer in cats in which FcaPV-2 infection is already well established. However, these results justify future studies in which the vaccine is administered to younger cats prior to FcaPV-2 infection becoming fully established., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Murine norovirus (MNV) viral protein genome-linked (VPg) manipulates the cell cycle to induce a G0/G1 arrest and gain a beneficial replication environment. All viruses of the norovirus genus encode a VPg protein; however, it is unknown if the G0/G1 arrest induced by MNV VPg is conserved in other members of the genus. RNA transcripts encoding a representative viral VPg from five norovirus genogroups were transfected into RAW-Blue murine macrophages, and the percentage of cells in each phase of the cell cycle was determined. A G0/G1 cell cycle arrest was observed for all norovirus VPg proteins tested, and in the wider Caliciviridae family the arrest was also conserved in rabbit hemorrhagic disease virus (RHDV) VPg and human sapovirus (HuSV) VPg. Truncation of MNV VPg shows that the first 62 amino acids are sufficient for a cell cycle arrest, and alignment of VPg sequences revealed a conserved motif in the N-terminal region of VPg. Analysis of VPg constructs with single N-terminal region point mutations, or exchange of N-terminal regions between VPg proteins, confirmed the importance of the N-terminal region for cell cycle arrest. These results provide evidence that G0/G1 cell cycle arrest is a conserved function of norovirus VPg proteins that involves the N-terminal region of these proteins.

Virus-like particles (VLP) from the rabbit haemorrhagic disease virus (RHDV) can deliver tumour antigens to induce anticancer immune responses. In this study, we explored how RHDV VLP can be functionalised to enhance the immune response by increasing antigen loading, incorporating linkers to enhance epitope processing, and targeting receptor-mediated internalisation of VLP. RHDV VLP were developed to deliver up to three copies of gp100 25-33 which contained proteasome cleavable linkers to target the correct processing of the epitope. Addition of mono- and dimannosides, conjugated to the surface of the gp100 VLP, would utilise a second pathway of internalisation, mannose receptor mediated, to further augment antigen internalised by phagocytosis/macropinocytosis. In vitro cell culture studies showed that a processing linker at the C-terminus of the epitope (gp100.1LC) induced enhanced T-cell activation (7.3 ng/ml interferon- (IFN-) γ release) compared to no linker (3.0 ng/ml IFN- γ ) or the linker at the N-terminus (0.8 ng/ml IFN- γ ). VLP delivering two (gp100.2L) or three (gp100.3L) gp100 epitopes induced similar high T-cell activation (7.6 ng/ml IFN- γ ) compared to gp100.1LC. An in vivo cytotoxicity assay and a therapeutic tumour trial confirmed that mice vaccinated with either gp100.2L or gp100.3L induced a specific antitumour immune response. Mannosylation of the gp100.2L VLP further enhanced the generated immune response, demonstrated by prolonged survival of mice vaccinated with dimannosylated gp100.2L VLP (D-gp100.2L) by 22 days compared to gp100.2L-vaccinated mice. This study showed that functionalisation of RHDV VLP by addition of an epitope-processing linker and mannosylation of the surface facilitates the efficacy of VLP as vaccination vectors for tumour immunotherapy.

Introduction: Virus-like particle (VLP) vaccines face significant challenges in their translation from laboratory models, to routine clinical administration. While some VLP vaccines thrive and are readily adopted into the vaccination schedule, others are restrained by regulatory obstacles, proprietary limitations, or finding their niche amongst the crowded vaccine market. Often the necessity to supplant an existing vaccination regimen possesses an immediate obstacle for the development of a VLP vaccine, despite any preclinical advantages identified over the competition. Novelty, adaptability and formulation compatibility may prove invaluable in helping place VLP vaccines at the forefront of vaccination technology., Areas Covered: The purpose of this review is to outline the diversity of VLP vaccines, VLP-specific immune responses, and to explore how modern formulation and delivery techniques can enhance the clinical relevance and overall success of VLP vaccines., Expert Commentary: The role of formation science, with an emphasis on the diversity of immune responses induced by VLP, is underrepresented amongst clinical trials for VLP vaccines. Harnessing such diversity, particularly through the use of combinations of select excipients and adjuvants, will be paramount in the development of VLP vaccines.

Background: Colorectal cancer is responsible for almost 700,000 deaths annually worldwide. Therapeutic vaccination is a promising alternative to conventional treatment for colorectal cancer, using vaccines to induce targeted immune responses against tumour-associated antigens. In this study, we have developed chimaeric virus-like particles (VLP), a form of non-infectious non-replicative subunit vaccine consisting of rabbit haemorrhagic disease virus (RHDV) VP60 capsid proteins containing recombinantly inserted epitopes from murine topoisomerase IIα and survivin. These vaccines were developed in mono- (T.VP60, S.VP60) and multi-target (TS.VP60) forms, aiming to elucidate the potential benefits from multi-target vaccination., Methods: Chimaeric RHDV VLP were developed by recombinantly inserting immune epitopes at the N-terminus of VP60. Vaccines were tested against a murine model of colorectal cancer by establishing MC38-OVA tumours subcutaneously. Unmethylated CpG DNA oligonucleotides (CpGs) were used as a vaccine adjuvant. Statistical tests employed included the Mantel-Cox log-rank test, ANOVA and unpaired t-tests depending on the data analysed, with a post hoc Bonferroni adjustment for multiple measures., Results: Chimaeric RHDV VLP were found to form a composite particle in the presence of CpGs. Overall survival was significantly improved amongst mice bearing MC38-OVA tumours following vaccination with T.VP60 (60%, 9/15), S.VP60 (60%, 9/15) or TS.VP60 (73%, 11/15). TS.VP60 significantly prolonged the vaccine-induced remission period in comparison to each mono-therapy., Conclusions: Chimaeric VLP containing multiple epitopes were found to confer an advantage for therapeutic vaccination in a model of colorectal cancer based on the prolongation of remission prior to tumour escape.

The Czech v351 strain of rabbit hemorrhagic disease virus (RHDV1) is used in Australia and New Zealand as a biological control agent for rabbits, which are important and damaging introduced vertebrate pests in these countries. However, nonpathogenic rabbit caliciviruses (RCVs) can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with effective rabbit biocontrol. Antibodies that cross-reacted against RHDV antigens were found in wild rabbits before the release of RHDV1 in New Zealand in 1997, suggesting that nonpathogenic RCVs were already present in New Zealand. The aim of this study was to confirm the presence of nonpathogenic RCV in New Zealand and describe its geographical distribution. RCV and RHDV antibody assays were used to screen serum samples from 350 wild rabbits from 14 locations in New Zealand. The serological survey indicated that both RCV and RHDV are widespread in New Zealand wild rabbits, with antibodies detected in 10 out of 14 and 12 out of 14 populations, respectively. Two closely related RCV strains were identified in the duodenal tissue from a New Zealand wild rabbit (RCV Gore-425A and RCV Gore-425B). Both variants are most closely related to Australian RCV strains, but with 88% nucleotide identity, they are genetically distinct. Phylogenetic analysis revealed that the New Zealand RCV strains fall within the genetic diversity of the Australian RCV isolates, indicating a relatively recent movement of RCVs between Australia and New Zealand. IMPORTANCE Wild rabbits are important and damaging introduced vertebrate pests in Australia and New Zealand. Although RHDV1 is used as a biological control agent, some nonpathogenic RCVs can provide partial immunological cross-protection against lethal RHDV infection and thus interfere with its effectiveness for rabbit control. The presence of nonpathogenic RCVs in New Zealand wild rabbits has been long hypothesized, but earlier attempts to isolate a New Zealand RCV strain have been unsuccessful. Therefore, it is important to determine if such nonpathogenic viruses exist in New Zealand rabbits, especially considering the proposed introduction of new RHDV strains into New Zealand as biocontrols., (Copyright © 2017 American Society for Microbiology.)

Background: The GII.4 Sydney 2012 strain of human norovirus (HuNoV) is a pandemic strain that is responsible for the majority of norovirus outbreaks in healthcare settings. The function of the non-structural (NS)1-2 protein from HuNoV is unknown., Results: In silico analysis of human norovirus NS1-2 protein showed that it shares features with the murine NS1-2 protein, including a disordered region, a transmembrane domain and H-box and NC sequence motifs. The proteins also contain caspase cleavage and phosphorylation sites, indicating that processing and phosphorylation may be a conserved feature of norovirus NS1-2 proteins. In this study, RNA transcripts of human and murine norovirus full-length and the disordered region of NS1-2 were transfected into monocytes, and next generation sequencing was used to analyse the transcriptomic profile of cells expressing virus proteins. The profiles were then compared to the transcriptomic profile of MNV-infected cells., Conclusions: RNAseq analysis showed that NS1-2 proteins from human and murine noroviruses affect multiple immune systems (chemokine, cytokine, and Toll-like receptor signaling) and intracellular pathways (NFκB, MAPK, PI3K-Akt signaling) in murine monocytes. Comparison to the transcriptomic profile of MNV-infected cells indicated the pathways that NS1-2 may affect during norovirus infection.

Unlabelled: Two closely related caliciviruses cocirculate in Australia: rabbit hemorrhagic disease virus (RHDV) and rabbit calicivirus Australia 1 (RCV-A1). RCV-A1 causes benign enteric infections in the European rabbit (Oryctolagus cuniculus) in Australia and New Zealand, while its close relative RHDV causes a highly pathogenic infection of the liver in the same host. The comparison of these viruses provides important information on the nature and trajectory of virulence evolution, particularly as highly virulent strains of RHDV may have evolved from nonpathogenic ancestors such as RCV-A1. To determine the evolution of RCV-A1 we sequenced the full-length genomes of 44 RCV-A1 samples isolated from healthy rabbits and compared key evolutionary parameters to those of its virulent relative, RHDV. Despite their marked differences in pathogenicity and tissue tropism, RCV-A1 and RHDV have evolved in a very similar manner. Both viruses have evolved at broadly similar rates, suggesting that their dynamics are largely shaped by high background mutation rates, and both exhibit occasional recombination and an evolutionary environment dominated by purifying selection. In addition, our comparative analysis revealed that there have been multiple changes in both virulence and tissue tropism in the evolutionary history of these and related viruses. Finally, these new genomic data suggest that either RCV-A1 was introduced into Australia after the introduction of myxoma virus as a biocontrol agent in 1950 or there was drastic reduction of the rabbit population, and hence of RCV-A1 genetic diversity, perhaps coincident with the emergence of myxoma virus., Importance: The comparison of closely related viruses that differ profoundly in propensity to cause disease in their hosts offers a powerful opportunity to reveal the causes of changes in virulence and to study how such changes alter the evolutionary dynamics of these pathogens. Here we describe such a novel comparison involving two closely related RNA viruses that cocirculate in Australia, the highly virulent rabbit hemorrhagic disease virus (RHDV) and the nonpathogenic rabbit calicivirus Australia 1 (RCV-A1). Both viruses infect the European rabbit, but they differ in virulence, tissue tropism, and mechanisms of transmission. Surprisingly, and despite these fundamental differences, RCV-A1 and RHDV have evolved at very similar (high) rates and with strong purifying selection. Furthermore, candidate key mutations were identified that may play a role in virulence and/or tissue tropism and therefore warrant further investigation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Murine norovirus-1 (MNV-1) is known to subvert host cell division inducing an accumulation of cells in the G0/G1 phase, creating conditions where viral replication is favored. This study identified that NS5 (VPg), is capable of inducing cell cycle arrest in the absence of viral replication or other viral proteins in an analogous manner to MNV-1 infection. NS5 expression induced an accumulation of cells in the G0/G1 phase in an asynchronous population by inhibiting progression at the G1/S restriction point. Furthermore, NS5 expression resulted in a down-regulation of cyclin A expression in asynchronous cells and inhibited cyclin A expression in cells progressing from G1 to S phase. The activity of NS5 on the host cell cycle occurs through an uncharacterized function. Amino acid substitutions of NS5(Y26A) and NS5(F123A) that inhibit the ability for NS5 to attach to RNA and recruit host eukaryotic translation initiation factors, respectively, retained the ability to induce an accumulation of cells in the G0/G1 phase as identified for wild-type NS5. To the best of our knowledge, this is the first report of a VPg protein manipulating the host cell cycle., Competing Interests: The authors have declared that no competing interests exist.

Activated antigen-presenting cells (APC) deliver the three signals cytotoxic T cells require to differentiate into effector cells that destroy the tumor. These comprise antigen, co-stimulatory signals and cytokines. Once these cells have carried out their function, they apoptose. We hypothesized that the tumor suppressor protein, p53, played an important role in generating the antitumor response facilitated by APC. CD11c + APC derived from p53 wild-type (wt) mouse (wt p53) GM-CSF bone marrow cultures (BMAPC) and activated had reduced survival compared to BMAPC from p53 null consistent with p53-mediated apoptosis following activation. There was a lower percentage of antigenic peptide/MHC I complexes on antigen-pulsed p53 null cells suggesting p53 played a role in antigen processing but there was no difference in antigen-specific T cell proliferative responses to these cells in vivo . In contrast, antigen-specific cytotoxicity in vivo was markedly reduced in response to p53 null BMAPC. When these cells were pulsed with a model tumor antigen and delivered as a prophylactic vaccination, they provided no protection against melanoma cell growth whereas wt BMAPC were very effective. This suggested that p53 might regulate the requisite third signal and, indeed, we found that p53 null BMAPC produced less IL-12 than wt p53 BMAPC and that p53 bound to the promoter region of IL-12. This work suggests that p53 in activated BMAPC is associated with the generation of IL-12 required for the differentiation of cytotoxic immune responses and an effective antitumor response. This is a completely new role for this protein that has implications for BMAPC-mediated immunotherapy.

Unlabelled: Many viruses replicate most efficiently in specific phases of the cell cycle, establishing or exploiting favorable conditions for viral replication, although little is known about the relationship between caliciviruses and the cell cycle. Microarray and Western blot analysis of murine norovirus 1 (MNV-1)-infected cells showed changes in cyclin transcript and protein levels indicative of a G1 phase arrest. Cell cycle analysis confirmed that MNV-1 infection caused a prolonging of the G1 phase and an accumulation of cells in the G0/G1 phase. The accumulation in G0/G1 phase was caused by a reduction in cell cycle progression through the G1/S restriction point, with MNV-1-infected cells released from a G1 arrest showing reduced cell cycle progression compared to mock-infected cells. MNV-1 replication was compared in populations of cells synchronized into specific cell cycle phases and in asynchronously growing cells. Cells actively progressing through the G1 phase had a 2-fold or higher increase in virus progeny and capsid protein expression over cells in other phases of the cell cycle or in unsynchronized populations. These findings suggest that MNV-1 infection leads to prolonging of the G1 phase and a reduction in S phase entry in host cells, establishing favorable conditions for viral protein production and viral replication. There is limited information on the interactions between noroviruses and the cell cycle, and this observation of increased replication in the G1 phase may be representative of other members of the Caliciviridae., Importance: Noroviruses have proven recalcitrant to growth in cell culture, limiting our understanding of the interaction between these viruses and the infected cell. In this study, we used the cell-culturable MNV-1 to show that infection of murine macrophages affects the G1/S cell cycle phase transition, leading to an arrest in cell cycle progression and an accumulation of cells in the G0/G1 phase. Furthermore, we show that MNV replication is enhanced in the G1 phase compared to other stages of the cell cycle. Manipulating the cell cycle or adapting to cell cycle responses of the host cell is a mechanism to enhance virus replication. To the best of our knowledge, this is the first report of a norovirus interacting with the host cell cycle and exploiting the favorable conditions of the G0/G1 phase for RNA virus replication., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitin-rich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides.

The norovirus NS6 protease is a key target for anti-viral drug development. Noroviruses encode a 2200 amino acid polyprotein which is cleaved by this critical protease at five defined boundary substrates into six mature non-structural (NS) proteins. Studies of the human norovirus (HNV) NS6 protease, in the context of a full ORF1 polyprotein, have been severely hampered because HNVs are not culturable. Thus, investigations into the HNV NS6 protease have been largely restricted to in vitro assays using Escherichia coli-expressed, purified enzyme. The NS6 protease is formed of two distinct domains joined by a linking loop. Structural data suggest that domain 2 of the protease possesses substantial substrate binding pockets which form the bulk of the interactions with the NS boundaries and largely dictate boundary specificity and cleavage. We have constructed chimaeric murine norovirus (MNV) genomes carrying individual domains from the HNV protease and demonstrated by cell transfection that chimaeric HNV proteases have functional activity in the context of the full-length ORF1 polyprotein. Although domain 2 primarily confers boundary specificity, our data suggest that an inter-domain interaction exists within HNV NS6 protease which influences cleavage of specific substrates. The present study also shows that chimaeric MNVs provide improved models for studying HNV protein function in the context of a full ORF1 polyprotein.

Virus-like particles (VLPs) are an effective means of establishing both prophylactic and therapeutic immunity against their source virus or heterologous antigens. The particulate nature and repetitive structure of VLPs makes them ideal for stimulating potent immune responses. Epitopes delivered by VLPs can be presented on MHC-II for stimulation of a humoral immune response, or cross-presented onto MHC-I leading to cell-mediated immunity. VLPs as particulate subunit vaccine carriers are showing promise in preclinical and clinical trials for the treatment of many conditions including cancer, autoimmunity, allergies and addiction. Supporting the delivery of almost any form of antigenic material, VLPs are ideal candidate vectors for development of future vaccines.

Internalization of peptides by antigen presenting cells is crucial for the initiation of the adaptive immune response. Mannosylation has been demonstrated to enhance antigen uptake through mannose receptors, leading to improved immune responses. In this study we test the effect of surface mannosylation of protein-based virus-like particles (VLP) derived from Rabbit hemorrhagic disease virus (RHDV) on uptake by murine and human antigen presenting cells. A monomannoside and a novel dimannoside were synthesized and successfully conjugated to RHDV VLP capsid protein, providing approximately 270 mannose groups on the surface of each virus particle. VLP conjugated to the mannoside or dimannoside exhibited significantly enhanced binding and internalization by murine dendritic cells, macrophages and B cells as well as human dendritic cells and macrophages. This uptake was inhibited by the inclusion of mannan as a specific inhibitor of mannose specific uptake, demonstrating that mannosylation of VLP targets mannose receptor-based uptake. Consistent with mannose receptor-based uptake, partial retargeting of the intracellular processing of RHDV VLP was observed, confirming that mannosylation of VLP provides both enhanced uptake and modified processing of associated antigens.

Noroviruses are an emerging threat to public health, causing large health and economic costs, including at least 200,000 deaths annually. The inability to replicate in cell culture or small animal models has limited the understanding of the interaction between human noroviruses and their hosts. However, an alternative strategy to gain insights into norovirus pathogenesis is to study murine norovirus (MNV-1) that replicates in cultured macrophages. While the innate immune response is central to the resolution of norovirus disease, the adaptive immune response is required for viral clearance. The specific responses of macrophages and dendritic cells to infection drive the adaptive immune response, with chemokines playing an important role. In this study, we have conducted microarray analysis of RAW264.7 macrophages infected with MNV-1 and examined the changes in chemokine transcriptional expression during infection. While the majority of chemokines showed no change, there was specific up-regulation in chemokines reflective of a bias toward a Th1 response, specifically CCL2, CCL3, CCL4, CCL5, CXCL2, CXCL10 and CXCL11. These changes in gene expression were reflected in protein levels as determined by ELISA assay. This virus-induced chemokine response will affect the resolution of infection and may limit the humoral response to norovirus infection., (Copyright © 2014. Published by Elsevier B.V.)

Investigations into human norovirus infection, replication and pathogenesis, as well as the development of potential antiviral agents, have been restricted by the lack of a cell culture system for human norovirus. To date, the optimal cell culture surrogate virus model for studying human norovirus biology is the murine norovirus (MNV). In this report we generate a tetracycline-regulated, inducible eukaryotic cell system expressing the entire MNV ORF1 polyprotein. Once induced, the MNV ORF1 polyprotein was faithfully processed to the six mature non-structural proteins that predominately located to a discrete perinuclear region, as has been observed in active MNV infection. Furthermore, we found that expression of the ORF1 polyprotein alone was sufficient to induce apoptosis, characterised by caspase-9 activation and survivin down-regulation. This cell line provides a valuable new tool for studying MNV ORF1 non-structural protein function, screening for potential antiviral agents and acts as a proof-of-principle for such systems to be developed for human noroviruses.

Previous studies suggest physical activity improves cognition and lowers Alzheimer's disease (AD) risk. However, key AD pathogenic factors that are thought to be influenced by physical activity, particularly plasma amyloid-β (Aβ) and Aβ brain load, have yet to be thoroughly investigated. The objective of this study was to determine if plasma Aβ and amyloid brain deposition are associated with physical activity levels, and whether these associations differed between carriers and non-carriers of the apolipoprotein E (APOE) ε4 allele. Five-hundred and forty six cognitively intact participants (aged 60-95 years) from the Australian Imaging, Biomarkers and Lifestyle Study of Ageing (AIBL) were included in these analyses. Habitual physical activity levels were measured using the International Physical Activity Questionnaire (IPAQ). Serum insulin, glucose, cholesterol and plasma Aβ levels were measured in fasting blood samples. A subgroup (n=116) underwent (11)C-Pittsburgh compound B (PiB) positron emission tomography (PET) scanning to quantify brain amyloid load. Higher levels of physical activity were associated with higher high density lipoprotein (HDL) (P=0.037), and lower insulin (P<0.001), triglycerides (P=0.019) and Aβ1-42/1-40 ratio (P=0.001). After stratification of the cohort based on APOE ε4 allele carriage, it was evident that only non-carriers received the benefit of reduced plasma Aβ from physical activity. Conversely, lower levels of PiB SUVR (standardised uptake value ratio) were observed in higher exercising APOE ε4 carriers. Lower plasma Aβ1-42/1-40 and brain amyloid was observed in those reporting higher levels of physical activity, consistent with the hypothesis that physical activity may be involved in the modulation of pathogenic changes associated with AD.

Virus-like particles (VLP) from rabbit hemorrhagic disease virus (RHDV) can be used as a scaffold to facilitate the delivery of antigens to induce cell-mediated immune responses. In this study, we investigated the immune response to lymphocytic choriomeningitis virus-derived peptide antigen (gp33) delivered by RHDV VLP. The gp33 peptides were incorporated into the VLP in 2 different forms, either recombinantly expressed inside the VLP (VLP-gp33r) or chemically coupled to the surface of the VLP (VLP-gp33c). We showed that VLP-gp33r induced a greater level of cytotoxicity than VLP-gp33c against gp33-coated target cells in vivo. Both VLP, when delivered as prophylactic vaccines, inhibited the growth of Lewis' lung carcinoma tumors expressing gp33 (LL-LCMV) in mice to a similar degree. Studies to investigate the mechanism induced by these VLP showed that 2 CD11c DC subsets, CD8α and CD8α, acquired VLP in vivo and in vitro, and VLP-gp33r were cross-presented by both these subsets to prime CD8 T cells through a TAP-independent, endosomal recycling pathway. Depletion of Langerin DC in vivo before and after vaccination with VLP-gp33r, lead to reduced cytotoxicity implicating these cells in the induction of cytotoxic effector cells. These results suggest that recombinant VLP expressing tumor peptides targeted to Langerin DC may have clinical application. Finally we found that VLP-gp33r were more effective antitumor vaccines than VLP-gp33c when delivered therapeutically. The findings of this study suggest the potential of VLP as a platform for delivery of tumor-associate antigen and elicit protective immunity against tumors.

The Mediterranean diet (MeDi), due to its correlation with a low morbidity and mortality for many chronic diseases, has been widely recognised as a healthy eating model. We aimed to investigate, in a cross-sectional study, the association between adherence to a MeDi and risk for Alzheimer's disease (AD) and mild cognitive impairment (MCI) in a large, elderly, Australian cohort. Subjects in the Australian Imaging, Biomarkers and Lifestyle Study of Ageing cohort (723 healthy controls (HC), 98 MCI and 149 AD participants) completed the Cancer Council of Victoria Food Frequency Questionnaire. Adherence to the MeDi (0- to 9-point scale with higher scores indicating higher adherence) was the main predictor of AD and MCI status in multinominal logistic regression models that were adjusted for cohort age, sex, country of birth, education, apolipoprotein E genotype, total caloric intake, current smoking status, body mass index, history of diabetes, hypertension, angina, heart attack and stroke. There was a significant difference in adherence to the MeDi between HC and AD subjects (P < 0.001), and in adherence between HC and MCI subjects (P < 0.05). MeDi is associated with change in Mini-Mental State Examination score over an 18-month time period (P < 0.05) in HCs. We conclude that in this Australian cohort, AD and MCI participants had a lower adherence to the MeDi than HC participants.

Virus-like particles (VLP) are effective vehicles for delivery of heterologous antigen to antigen-presenting cells. However VLP alone are insufficiently stimulatory to generate the signals required to facilitate effective priming of naïve T cells. We show that the VLP derived from rabbit hemorrhagic disease virus can bind the galactose-containing adjuvant α-galactosylceramide to form a composite particle for co-delivery of antigen and adjuvant to the same antigen-presenting cell. Vaccination with VLP and α-galactosylceramide activated splenic iNKT cells to produce IFN-γ and IL-4, led to the generation of antigen-specific T cells that protected prophylactically against subcutaneous tumor challenge, and was more effective at generating anti-tumor immune responses than either component individually. These data demonstrate a novel method for immunopotentiating VLP to increase their efficacy in the generation of anti-tumor responses via the innate ligand recognition properties of calicivirus-derived nanoparticles., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Background: Tumour cell lysates are an excellent source of many defined and undefined tumour antigens and have been used clinically in immunotherapeutic regimes but with limited success., Methods: We conjugated Mel888 melanoma lysates to rabbit haemorrhagic disease virus virus-like particles (VLP), which can act as vehicles to deliver multiple tumour epitopes to dendritic cells (DC) to effectively activate antitumour responses., Results: Virus-like particles did not stimulate the phenotypic maturation of DC although, the conjugation of lysates to VLP (VLP-lysate) did overcome lysate-induced suppression of DC activation. Lysate-conjugated VLP enhanced delivery of antigenic proteins to DC, while the co-delivery of VLP-lysates with OK432 resulted in cross-priming of naïve T cells, with expansion of a MART1(+) population of CD8(+) T cells and generation of a specific cytotoxic response against Mel888 tumour cell targets. The responses generated with VLP-lysate and OK432 were superior to those stimulated by unconjugated lysate with OK432., Conclusion: Collectively, these results show that the combination of VLP-lysate with OK432 delivered to DC overcomes the suppressive effects of lysates, and enables priming of naïve T cells with superior ability to specifically kill their target tumour cells.

Human noroviruses are highly infectious viruses that cause the majority of acute, non-bacterial epidemic gastroenteritis cases worldwide. The first open reading frame of the norovirus RNA genome encodes for a polyprotein that is cleaved by the viral protease into six non-structural proteins. The first non-structural protein, NS1-2, lacks any significant sequence similarity to other viral or cellular proteins and limited information is available about the function and biophysical characteristics of this protein. Bioinformatic analyses identified an inherently disordered region (residues 1-142) in the highly divergent N-terminal region of the norovirus NS1-2 protein. Expression and purification of the NS1-2 protein of Murine norovirus confirmed these predictions by identifying several features typical of an inherently disordered protein. These were a biased amino acid composition with enrichment in the disorder promoting residues serine and proline, a lack of predicted secondary structure, a hydrophilic nature, an aberrant electrophoretic migration, an increased Stokes radius similar to that predicted for a protein from the pre-molten globule family, a high sensitivity to thermolysin proteolysis and a circular dichroism spectrum typical of an inherently disordered protein. The purification of the NS1-2 protein also identified the presence of an NS1-2 dimer in Escherichia coli and transfected HEK293T cells. Inherent disorder provides significant advantages including structural flexibility and the ability to bind to numerous targets allowing a single protein to have multiple functions. These advantages combined with the potential functional advantages of multimerisation suggest a multi-functional role for the NS1-2 protein.

Iridoviruses (IV) are nuclear cytoplasmic large DNA viruses that are receiving increasing attention as sublethal pathogens of a range of insects. Invertebrate iridovirus type 9 (IIV-9; Wiseana iridovirus) is a member of the major phylogenetic group of iridoviruses for which there is very limited genomic and proteomic information. The genome is 205,791 bp, has a G+C content of 31%, and contains 191 predicted genes, with approximately 20% of its repeat sequences being located predominantly within coding regions. The repeated sequences include 11 proteins with helix-turn-helix motifs and genes encoding related tandem repeat amino acid sequences. Of the 191 proteins encoded by IIV-9, 108 are most closely related to orthologs in IIV-3 (Chloriridovirus genus), and 114 of the 126 IIV-3 genes have orthologs in IIV-9. In contrast, only 97 of 211 IIV-6 genes have orthologs in IIV-9. There is almost no conservation of gene order between IIV-3, IIV-6, and IIV-9. Phylogenetic analysis using a concatenated sequence of 26 core IV genes confirms that IIV-3 is more closely related to IIV-9 than to IIV-6, despite being from a different genus of the Iridoviridae. An interaction between IIV and small RNA regulatory systems is supported by the prediction of seven putative microRNA (miRNA) sequences combined with XRN exonuclease, RNase III, and double-stranded RNA binding activities encoded on the genome. Proteomic analysis of IIV-9 identified 64 proteins in the virus particle and, when combined with infected cell analysis, confirmed the expression of 94 viral proteins. This study provides the first full-genome and consequent proteomic analysis of group II IIV.

Effective vaccines and immunotherapies against cancer require professional antigen-presenting cells to cross-present exogenous antigen to initiate cytotoxic T-cell responses to destroy tumors. Virus-like particles (VLPs), containing tumor antigens, which can immunize against cancers, are cross-presented by dendritic cell (DC) but the mechanism by which this occurs is not fully understood. Here, we used VLPs, derived from rabbit hemorrhagic disease virus (RHDV) with both murine and human DCs, to elucidate these pathways. We have employed inhibitors to demonstrate that these VLPs are taken up by clathrin-dependent macropinocytosis and phagocytosis before being degraded in acidic lysosomal compartments. VLP-derived peptides are loaded onto major histocompatibility complex I that have been recycled from the cell surface. Antigen-coupled VLPs and murine ovalbumin-specific and human melanoma-associated antigen recognized by T cells (MART-1)-specific CD8(+) T cells were used to demonstrate cross-presentation via this alternate, receptor recycling pathway, which operated independently of the proteasome and the transporter-associated with antigen presentation. Finally, we found that cross-presentation of VLPs in vivo was not confined to CD8α(+) DC subsets. These data define the cross-presentation pathway for RHDV VLPs and may lead to improved cancer immunotherapies.

Our previous structural studies on intact, infectious murine norovirus 1 (MNV-1) virions demonstrated that the receptor binding protruding (P) domains are lifted off the inner shell of the virus. Here, the three-dimensional (3D) reconstructions of recombinant rabbit hemorrhagic disease virus (rRHDV) virus-like particles (VLPs) and intact MNV-1 were determined to approximately 8-A resolution. rRHDV also has a raised P domain, and therefore, this conformation is independent of infectivity and genus. The atomic structure of the MNV-1 P domain was used to interpret the MNV-1 reconstruction. Connections between the P and shell domains and between the floating P domains were modeled. This observed P-domain flexibility likely facilitates virus-host receptor interactions.

The lightbrown apple moth (Epiphyas postvittana) is a leafroller pest that damages horticultural crops in New Zealand. This paper documents the establishment of a primary cell line from neonate E. postvittana larvae to facilitate the development of E. postvittana nucleopolyhedrovirus (EppoNPV) for control of this pest. The cell line was cultured for 36 passages and a clonal derivative designated EpN1.10 was generated that had a doubling time of 36h at 21 degrees C. The EpN1.10 cell line allowed for recovery of EppoNPV from transfected genomic DNA and virus passage, as determined by occlusion body production and restriction endonuclease analysis.

The Tetraviridae is a family of non-enveloped positive-stranded RNA insect viruses that is defined by the T=4 symmetry of virions. We report the complete Euprosterna elaeasa virus (EeV) genome sequence of 5698 nt with no poly(A) tail and two overlapping open reading frames, encoding the replicase and capsid precursor, with approximately 67% amino acid identity to Thosea asigna virus (TaV). The N-terminally positioned 17 kDa protein is released from the capsid precursor by a NPGP motif. EeV has 40 nm non-enveloped isometric particles composed of 58 and 7 kDa proteins. The 3'-end of TaV/EeV is predicted to form a conserved pseudoknot. Replicases of TaV and EeV include a newly delineated VPg signal mediating the protein priming of RNA synthesis in dsRNA Birnaviridae. Results of rooted phylogenetic analysis of replicase and capsid proteins are presented to implicate recombination between monopartite tetraviruses, involving autonomization of a sgRNA, in the emergence of bipartite tetraviruses. They are also used to revise the Tetraviridae taxonomy., (Copyright 2009 Elsevier Inc. All rights reserved.)

Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the N-terminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be alpha-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-A structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.

Host cell and virus gene expression were measured five days after per os inoculation of 3rd instar lightbrown apple moth (LBAM) larvae with the Epiphyas postvittana nucleopolyhedrovirus (EppoNPV). Microarray analysis identified 84 insect genes that were up-regulated and 18 genes that were down-regulated in virus-infected larvae compared with uninfected larvae. From the 134 viral open reading frames represented on the microarray, 81 genes showed strong expression. Of the 38 functionally identifiable regulated insect genes, 23 coded for proteins that have roles in one of five processes; regulation of transcription and translation, induction of apoptosis, and maintenance of both juvenility and actin cytoskeletal integrity. Of the 34 functionally identifiable viral genes that were most strongly expressed, 12 had functions associated with these five processes, as did a further seven viral genes which were expressed at slightly lower levels. A survey of the LBAM-expressed sequence tag library identified further genes involved in these processes. In total, 135 insect genes and 38 viral genes were analysed by quantitative polymerase chain reaction. Twenty-one insect genes were strongly up-regulated and 31 genes strongly down-regulated. All 38 viral genes examined were highly expressed. These data suggest that induction of apoptosis and regulation of juvenility are the major 'battlegrounds' between virus and insect, with the majority of changes observed representing viral control of insect gene expression. Transcription and translational effects seem to be exerted largely through modulation of mRNA and protein degradation. Examples of attempts by the insect to repel the infection via changes in gene expression within these same processes were, however, also noted. The data also showed the extent to which viral transcription dominated in the infected insects at five days post inoculation.

Recombinant virus-like particles (VLP) expressing heterologous tumor antigens have recently been investigated for use as vaccines. We have chemically conjugated ovalbumin (OVA) or OVA-derived CD4 (OTII) and CD8 (OTI) epitopes, to rabbit hemorrhagic disease virus (RHDV) VLP. VLP conjugated with OVA were able to cross-prime CD8+ cells from OT1 mice transgenic for the OVA T cell receptor. VLP.OTI was able to induce higher antigen-specific cytotoxicity in vivo than VLP mixed with either the protein or the peptide. Furthermore we have shown that the growth of the aggressive B16.OVA melanoma in mice was significantly delayed in those animals that had been vaccinated with VLP.OVA or with VLP coupled with both OTI and OTII peptides prior to the introduction of the tumor. Neither VLP.OTI nor VLP.OTII alone were capable of inhibiting tumor growth. This work suggests that RHDV VLP offer a versatile scaffold for multiple vaccine epitopes, enabling cross-presentation of the antigen to elicit potent cell-mediated and anti-tumor responses.

The yield of two proteins, avidin and green fluorescent protein (GFP), expressed from a modified Autographa californica nucleopolyhedrovirus (AcMNPV), was compared in Sf9 cell culture monolayer, Sf21 cell suspension culture and intact Spodoptera litura larvae. GFP expressed from the p10 promoter yielded up to 1.5% of total soluble protein in larvae, 20-fold higher than that in monolayer suspension culture. Avidin, expressed from the polh promoter, yielded up to 2.3% of total soluble protein in larvae, 10-fold higher than that in suspension culture and 40-fold higher than that in monolayers. Avidin expression did not affect amounts of GFP in dual-expressing baculovirus compared with those detected from a GFP-only expressing AcMNPV. A biotin-binding assay showed that all avidin expressed in larvae was fully active. Glycosylation patterns of chicken-avidin and Spodoptera-avidin were very similar, though the latter showed a proportion of partially glycosylated material.

Baculoviruses deliver strong activation signals to dendritic cells and can promote potent immune responses. These properties can be harnessed to use baculovirus as an adjuvant and carrier particle for immunogenic peptides. In this study we use a chemical linker to couple peptides to the baculovirus Autographa californica multicapsid nucleopolyhedrovirus (AcMNPV). Intranasal delivery of baculovirus coupled with immunogenic peptides to mice elicited antigen-specific IgG1 and IgG2a antibody. Furthermore, antigen-specific IgA was detected in the lung, and an IFN-gamma response was observed upon re-stimulation with antigen. We show that chemical coupling enables the rapid modification of AcMNPV, allowing multiple epitopes to be delivered simultaneously on a self-adjuvanting carrier particle.

Virus-like particles have proved to be excellent molecular scaffolds, yet the individual characteristics and immune responses generated against each VLP requires the development of a wide range of capsids for use as vaccines, molecular delivery vessels, and nanoscale templates. Here we describe the development of Rabbit haemorrhagic disease virus (RHDV)-like particles as a rapidly versatile molecular workbench, overcoming limitations imposed by established genetic antigen incorporation procedures with chimeric VLP. Production of the RHDV capsid protein in a baculovirus system led to the self-assembly of VLP which were recovered at over 99% purity and manipulated both genetically and chemically. Fusion of small peptide sequences to RHDV VLP was well tolerated, forming chimeric capsids that enhanced the presentation of foreign peptide to hybridoma T helper cells 700-fold. Rapid and simple conjugation techniques employing the hetero-bifunctional chemical linker sulfo-SMCC enabled both small peptides and whole proteins to be conjugated to the surface of RHDV VLP, overcoming limitations imposed on VLP formation and yield experienced with chimeric VLP. Administration of VLP/ovalbumin conjugate provoked high titre ovalbumin-specific antibody in mice, demonstrating the immune stimulatory properties of the capsid were conferred to conjugated foreign antigen. VLP facilitated delivery of conjugated antigen to dendritic cells, eliciting proliferative responses in naïve TCR transgenic T helper cells that were at least 10-fold greater than ovalbumin antigen delivered alone.