CRISPR activation as a platform to identify interferon stimulated genes with anti-viral function.

Interferon Stimulated Gene (ISG) expression plays a key role in the control of viral replication and development of a robust adaptive response. Understanding this dynamic relationship between the pathogen and host is critical to our understanding of viral life-cycles and development of potential novel anti-viral strategies. Traditionally, plasmid based exogenous prompter driven expression of ISGs has been used to investigate anti-viral ISG function, however there are deficiencies in this approach. To overcome this, we investigated the utility of CRISPR activation (CRISPRa), which allows for targeted transcriptional activation of a gene from its endogenous promoter. Using the CRISPRa-SAM system to induce targeted expression of a panel of anti-viral ISGs we showed robust induction of mRNA and protein expression. We then employed our CRISPRa-SAM ISG panel in several antiviral screen formats to test for the ability of ISGs to prevent viral induced cytopathic cell death (CPE) and replication of Dengue Virus (DENV), Zika Virus (ZIKV), West Nile Virus Kunjin (WNVKUN), Hepatitis A Virus (HAV) and Human Coronavirus 229E (HCoV-229E). Our CRISPRa approach confirmed the anti-viral activity of ISGs like IFI6, IFNß and IFNλ2 that prevented viral induced CPE, which was supported by high-content immunofluorescence imaging analysis. This work highlights CRISPRa as a rapid, agile, and powerful methodology to identify and characterise ISGs and viral restriction factors.

Assessment of hepatitis B virus infection and interhost cellular responses using intrahepatic cholangiocyte organoids.

The intrahepatic cholangiocyte organoids (ICOs) model was evaluated for host differences in hepatitis B virus (HBV) infection, cellular responses, antiviral and immunomodulator responses. Twelve ICOs generated from liver resections and biopsies were assessed for metabolic markers and functional HBV entry receptor expression throughout differentiation. Structural changes relevant to HBV infection were characterized using histology, confocal, and electron microscopy examinations. Optimal ICO culture conditions for HBV infection using HepAD38 (genotype D) and plasma-derived HBV (genotype B and C) were described. HBV infection was confirmed using HBcAg immunostaining, qRT-PCR (RNA, covalently closed circular DNA [cccDNA], extracellular DNA) and ELISA (HBsAg and HBeAg). Drug response to antiviral and immunosuppressive agent, and cellular responses (interferon-stimulated genes [ISG]) to interferon-α and viral mimic (PolyI:C) were assessed. ICOs underwent metabolic and structural remodeling following differentiation. Optimal HBV infection was achieved in well-differentiated ICOs using spinoculation, with time and donor-dependent increase in HBV RNA, cccDNA, extracellular DNA, HBeAg and HBsAg. Donor-dependent drug responsiveness to entry inhibitor and JAK inhibitor was observed. Despite having a robust ISG response to interferon-α and PolyI:C, HBV infection in ICOs did not upregulate ISGs. Human ICOs support HBV infection and replication with donor-dependent variation in viral dynamics and cellular responses. These features can be utilized for the development of personalized drug testing platform for antivirals.

NanoFIRE: A NanoLuciferase and Fluorescent Integrated Reporter Element for Robust and Sensitive Investigation of HIF and Other Signalling Pathways.

The Hypoxia Inducible Factor (HIF) transcription factors are imperative for cell adaption to low oxygen conditions and development; however, they also contribute to ischaemic disease and cancer. To identify novel genetic regulators which target the HIF pathway or small molecules for therapeutic use, cell-based reporter systems are commonly used. Here, we present a new, highly sensitive and versatile reporter system, NanoFIRE: a NanoLuciferase and Fluorescent Integrated Reporter Element. Under the control of a Hypoxic Response Element (HRE-NanoFIRE), this system is a robust sensor of HIF activity within cells and potently responds to both hypoxia and chemical inducers of the HIF pathway in a highly reproducible and sensitive manner, consistently achieving 20 to 150-fold induction across different cell types and a Z' score > 0.5. We demonstrate that the NanoFIRE system is adaptable via substitution of the response element controlling NanoLuciferase and show that it can report on the activity of the transcriptional regulator Factor Inhibiting HIF, and an unrelated transcription factor, the Progesterone Receptor. Furthermore, the lentivirus-mediated stable integration of NanoFIRE highlights the versatility of this system across a wide range of cell types, including primary cells. Together, these findings demonstrate that NanoFIRE is a robust reporter system for the investigation of HIF and other transcription factor-mediated signalling pathways in cells, with applications in high throughput screening for the identification of novel small molecule and genetic regulators.

Identification of Key Residues in Dengue Virus NS1 Protein That Are Essential for Its Secretion.

Dengue virus (DENV) non-structural protein 1 (NS1) is involved in multiple aspects of the DENV lifecycle. Importantly, it is secreted from infected cells as a hexameric lipoparticle that mediates vascular damage that is a hallmark of severe dengue. Although the secretion of NS1 is known to be important in DENV pathogenesis, the exact molecular features of NS1 that are required for its secretion from cells are not fully understood. In this study, we employed random point mutagenesis in the context of an NS1 expression vector encoding a C-terminal HiBiT luminescent peptide tag to identify residues within NS1 that are essential for its secretion. Using this approach, we identified 10 point mutations that corresponded with impaired NS1 secretion, with in silico analyses indicating that the majority of these mutations are located within the ß-ladder domain. Additional studies on two of these mutants, V220D and A248V, revealed that they prevented viral RNA replication, while studies using a DENV NS1-NS5 viral polyprotein expression system demonstrated that these mutations resulted in a more reticular NS1 localisation pattern and failure to detect mature NS1 at its predicted molecular weight by Western blotting using a conformation-specific monoclonal antibody. Together, these studies demonstrate that the combination of a luminescent peptide tagged NS1 expression system with random point mutagenesis enables rapid identification of mutations that alter NS1 secretion. Two such mutations identified via this approach revealed residues that are essential for correct NS1 processing or maturation and viral RNA replication.

Constitutive expression and distinct properties of IFN-epsilon protect the female reproductive tract from Zika virus infection.

The immunological surveillance factors controlling vulnerability of the female reproductive tract (FRT) to sexually transmitted viral infections are not well understood. Interferon-epsilon (IFNɛ) is a distinct, immunoregulatory type-I IFN that is constitutively expressed by FRT epithelium and is not induced by pathogens like other antiviral IFNs α, ß and λ. We show the necessity of IFNɛ for Zika Virus (ZIKV) protection by: increased susceptibility of IFNɛ-/- mice; their "rescue" by intravaginal recombinant IFNɛ treatment and blockade of protective endogenous IFNɛ by neutralising antibody. Complementary studies in human FRT cell lines showed IFNɛ had potent anti-ZIKV activity, associated with transcriptome responses similar to IFNλ but lacking the proinflammatory gene signature of IFNα. IFNɛ activated STAT1/2 pathways similar to IFNα and λ that were inhibited by ZIKV-encoded non-structural (NS) proteins, but not if IFNε exposure preceded infection. This scenario is provided by the constitutive expression of endogenous IFNε. However, the IFNɛ expression was not inhibited by ZIKV NS proteins despite their ability to antagonise the expression of IFNß or λ. Thus, the constitutive expression of IFNɛ provides cellular resistance to viral strategies of antagonism and maximises the antiviral activity of the FRT. These results show that the unique spatiotemporal properties of IFNε provides an innate immune surveillance network in the FRT that is a significant barrier to viral infection with important implications for prevention and therapy.

SARS-CoV-2 Omicron variant escapes neutralizing antibodies and T cell responses more efficiently than other variants in mild COVID-19 convalescents.

Coronavirus disease 2019 (COVID-19) convalescents living in regions with low vaccination rates rely on post-infection immunity for protection against re-infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We evaluate humoral and T cell immunity against five variants of concern (VOCs) in mild-COVID-19 convalescents at 12 months after infection with ancestral virus. In this cohort, ancestral, receptor-binding domain (RBD)-specific antibody and circulating memory B cell levels are conserved in most individuals, and yet serum neutralization against live B.1.1.529 (Omicron) is completely abrogated and significantly reduced for other VOCs. Likewise, ancestral SARS-CoV-2-specific memory T cell frequencies are maintained in >50% of convalescents, but the cytokine response in these cells to mutated spike epitopes corresponding to B.1.1.529 and B.1.351 (Beta) VOCs were impaired. These results indicate that increased antigen variability in VOCs impairs humoral and spike-specific T cell immunity post-infection, strongly suggesting that COVID-19 convalescents are vulnerable and at risk of re-infection with VOCs, thus stressing the importance of vaccination programs.

Long-term perturbation of the peripheral immune system months after SARS-CoV-2 infection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a highly infectious respiratory virus which is responsible for the coronavirus disease 2019 (COVID-19) pandemic. It is increasingly clear that recovered individuals, even those who had mild COVID-19, can suffer from persistent symptoms for many months after infection, a condition referred to as "long COVID", post-acute sequelae of COVID-19 (PASC), post-acute COVID-19 syndrome, or post COVID-19 condition. However, despite the plethora of research on COVID-19, relatively little is known about the molecular underpinnings of these long-term effects. METHODS: We have undertaken an integrated analysis of immune responses in blood at a transcriptional, cellular, and serological level at 12, 16, and 24 weeks post-infection (wpi) in 69 patients recovering from mild, moderate, severe, or critical COVID-19 in comparison to healthy uninfected controls. Twenty-one of these patients were referred to a long COVID clinic and > 50% reported ongoing symptoms more than 6 months post-infection. RESULTS: Anti-Spike and anti-RBD IgG responses were largely stable up to 24 wpi and correlated with disease severity. Deep immunophenotyping revealed significant differences in multiple innate (NK cells, LD neutrophils, CXCR3+ monocytes) and adaptive immune populations (T helper, T follicular helper, and regulatory T cells) in convalescent individuals compared to healthy controls, which were most strongly evident at 12 and 16 wpi. RNA sequencing revealed significant perturbations to gene expression in COVID-19 convalescents until at least 6 months post-infection. We also uncovered significant differences in the transcriptome at 24 wpi of convalescents who were referred to a long COVID clinic compared to those who were not. CONCLUSIONS: Variation in the rate of recovery from infection at a cellular and transcriptional level may explain the persistence of symptoms associated with long COVID in some individuals.

CRISPR Tackles Emerging Viral Pathogens.

Understanding the dynamic relationship between viral pathogens and cellular host factors is critical to furthering our knowledge of viral replication, disease mechanisms and development of anti-viral therapeutics. CRISPR genome editing technology has enhanced this understanding, by allowing identification of pro-viral and anti-viral cellular host factors for a wide range of viruses, most recently the cause of the COVID-19 pandemic, SARS-CoV-2. This review will discuss how CRISPR knockout and CRISPR activation genome-wide screening methods are a robust tool to investigate the viral life cycle and how other class 2 CRISPR systems are being repurposed for diagnostics.

Viperin is anti-viral in vitro but is dispensable for restricting dengue virus replication or induction of innate and inflammatory responses in vivo.

Viperin has antiviral function against many viruses, including dengue virus (DENV), when studied in cells in culture. Here, the antiviral actions of viperin were defined both in vitro and in a mouse in vivo model of DENV infection. Murine embryonic fibroblasts (MEFs) derived from mice lacking viperin (vip-/-) showed enhanced DENV infection, accompanied by increased IFN-ß and induction of ISGs; IFIT1 and CXCL-10 but not IRF7, when compared to wild-type (WT) MEFs. In contrast, subcutaneous challenge of immunocompetent WT and vip-/- mice with DENV did not result in enhanced infection. Intracranial infection with DENV resulted in body weight loss and neurological disease with a moderate increase in mortality in vip-/- compared with WT mice, although this was not accompanied by altered brain morphology, immune cell infiltration or DENV RNA level in the brain. Similarly, DENV induction of IFN-ß, IFIT1, CXCL-10, IRF7 and TNF-α was not significantly different in WT and vip-/- mouse brain, although there was a modest but significant increase in DENV induction of IL-6 and IfI27la in the absence of viperin. NanoString nCounter analysis confirmed no significant difference in induction of a panel of inflammatory genes in WT compared to vip-/- DENV-infected mouse brains. Further, polyI:C stimulation of bone marrow-derived macrophages (BMDMs) induced TNF-α, IFN-ß, IL-6 and Nos-2, but responses were not different in BMDMs generated from WT or vip-/- mice. Thus, while there is significant evidence of anti-DENV actions of viperin in some cell types in vitro, for DENV infection in vivo a lack of viperin does not affect systemic or brain susceptibility to DENV or induction of innate and inflammatory responses.

Genome-Wide CRISPR Screen Identifies RACK1 as a Critical Host Factor for Flavivirus Replication.

Cellular factors have important roles in all facets of the flavivirus replication cycle. Deciphering viral-host protein interactions is essential for understanding the flavivirus life cycle as well as development of effective antiviral strategies. To uncover novel host factors that are co-opted by multiple flaviviruses, a CRISPR/Cas9 genome wide knockout (KO) screen was employed to identify genes required for replication of Zika virus (ZIKV). Receptor for Activated Protein C Kinase 1 (RACK1) was identified as a novel host factor required for ZIKV replication, which was confirmed via complementary experiments. Depletion of RACK1 via siRNA demonstrated that RACK1 is important for replication of a wide range of mosquito- and tick-borne flaviviruses, including West Nile Virus (WNV), Dengue Virus (DENV), Powassan Virus (POWV) and Langat Virus (LGTV) as well as the coronavirus SARS-CoV-2, but not for YFV, EBOV, VSV or HSV. Notably, flavivirus replication was only abrogated when RACK1 expression was dampened prior to infection. Utilising a non-replicative flavivirus model, we show altered morphology of viral replication factories and reduced formation of vesicle packets (VPs) in cells lacking RACK1 expression. In addition, RACK1 interacted with NS1 protein from multiple flaviviruses; a key protein for replication complex formation. Overall, these findings reveal RACK1's crucial role to the biogenesis of pan-flavivirus replication organelles. IMPORTANCE Cellular factors are critical in all facets of viral lifecycles, where overlapping interactions between the virus and host can be exploited as possible avenues for the development of antiviral therapeutics. Using a genome-wide CRISPR knockout screening approach to identify novel cellular factors important for flavivirus replication we identified RACK1 as a pro-viral host factor for both mosquito- and tick-borne flaviviruses in addition to SARS-CoV-2. Using an innovative flavivirus protein expression system, we demonstrate for the first time the impact of the loss of RACK1 on the formation of viral replication factories known as 'vesicle packets' (VPs). In addition, we show that RACK1 can interact with numerous flavivirus NS1 proteins as a potential mechanism by which VP formation can be induced by the former.

Viperin interacts with PEX19 to mediate peroxisomal augmentation of the innate antiviral response.

Peroxisomes are recognized as significant platforms for the activation of antiviral innate immunity where stimulation of the key adapter molecule mitochondrial antiviral signaling protein (MAVS) within the RIG-I like receptor (RLR) pathway culminates in the up-regulation of hundreds of ISGs, some of which drive augmentation of multiple innate sensing pathways. However, whether ISGs can augment peroxisome-driven RLR signaling is currently unknown. Using a proteomics-based screening approach, we identified Pex19 as a binding partner of the ISG viperin. Viperin colocalized with numerous peroxisomal proteins and its interaction with Pex19 was in close association with lipid droplets, another emerging innate signaling platform. Augmentation of the RLR pathway by viperin was lost when Pex19 expression was reduced. Expression of organelle-specific MAVS demonstrated that viperin requires both mitochondria and peroxisome MAVS for optimal induction of IFN-ß. These results suggest that viperin is required to enhance the antiviral cellular response with a possible role to position the peroxisome at the mitochondrial/MAM MAVS signaling synapse, furthering our understanding of the importance of multiple organelles driving the innate immune response against viral infection.

Viperin binds STING and enhances the type-I interferon response following dsDNA detection.

Viperin is an interferon-inducible protein that is pivotal for eliciting an effective immune response against an array of diverse viral pathogens. Here we describe a mechanism of viperin's broad antiviral activity by demonstrating the protein's ability to synergistically enhance the innate immune dsDNA signaling pathway to limit viral infection. Viperin co-localized with the key signaling molecules of the innate immune dsDNA sensing pathway, STING and TBK1; binding directly to STING and inducing enhanced K63-linked polyubiquitination of TBK1. Subsequent analysis identified viperin's necessity to bind the cytosolic iron-sulfur assembly component 2A, to prolong its enhancement of the type-I interferon response to aberrant dsDNA. Here we show that viperin facilitates the formation of a signaling enhanceosome, to coordinate efficient signal transduction following activation of the dsDNA signaling pathway, which results in an enhanced antiviral state. We also provide evidence for viperin's radical SAM enzymatic activity to self-limit its immunomodulatory functions. These data further define viperin's role as a positive regulator of innate immune signaling, offering a mechanism of viperin's broad antiviral capacity.

The Molecular Interactions of ZIKV and DENV with the Type-I IFN Response.

Zika Virus (ZIKV) and Dengue Virus (DENV) are related viruses of the Flavivirus genus that cause significant disease in humans. Existing control measures have been ineffective at curbing the increasing global incidence of infection for both viruses and they are therefore prime targets for new vaccination strategies. Type-I interferon (IFN) responses are important in clearing viral infection and for generating efficient adaptive immune responses towards infection and vaccination. However, ZIKV and DENV have evolved multiple molecular mechanisms to evade type-I IFN production. This review covers the molecular interactions, from detection to evasion, of these viruses with the type-I IFN response. Additionally, we discuss how this knowledge can be exploited to improve the design of new vaccine strategies.

Sterility of gamma-irradiated pathogens: a new mathematical formula to calculate sterilizing doses.

In recent years there has been increasing advocacy for highly immunogenic gamma-irradiated vaccines, several of which are currently in clinical or pre-clinical trials. Importantly, various methods of mathematical modelling and sterility testing are employed to ensure sterility. However, these methods are designed for materials with a low bioburden, such as food and pharmaceuticals. Consequently, current methods may not be reliable or applicable to estimate the irradiation dose required to sterilize microbiological preparations for vaccine purposes, where bioburden is deliberately high. In this study we investigated the applicability of current methods to calculate the sterilizing doses for different microbes. We generated inactivation curves that demonstrate single-hit and multiple-hit kinetics under different irradiation temperatures for high-titre preparations of pathogens with different genomic structures. Our data demonstrate that inactivation of viruses such as Influenza A virus, Zika virus, Semliki Forest virus and Newcastle Disease virus show single-hit kinetics following exposure to gamma-irradiation. In contrast, rotavirus inactivation shows multiple-hit kinetics and the sterilizing dose could not be calculated using current mathematical methods. Similarly, Streptococcus pneumoniae demonstrates multiple-hit kinetics. These variations in killing curves reveal an important gap in current mathematical formulae to determine sterility assurance levels. Here we propose a simple method to calculate the irradiation dose required for a single log10 reduction in bioburden (D10) value and sterilizing doses, incorporating both single- and multiple-hit kinetics, and taking into account the possible existence of a resistance shoulder for some pathogens following exposure to gamma-irradiation.

Identification of Estrogen Receptor Modulators as Inhibitors of Flavivirus Infection.

Flaviviruses such as Zika virus (ZIKV), dengue virus (DENV), and West Nile virus (WNV) are major global pathogens for which safe and effective antiviral therapies are not currently available. To identify antiviral small molecules with well-characterized safety and bioavailability profiles, we screened a library of 2,907 approved drugs and pharmacologically active compounds for inhibitors of ZIKV infection using a high-throughput cell-based immunofluorescence assay. Interestingly, estrogen receptor modulators raloxifene hydrochloride and quinestrol were among 15 compounds that significantly inhibited ZIKV infection in repeat screens. Subsequent validation studies revealed that these drugs effectively inhibit ZIKV, DENV, and WNV (Kunjin strain) infection at low micromolar concentrations with minimal cytotoxicity in Huh-7.5 hepatoma cells and HTR-8 placental trophoblast cells. Since these cells lack detectable expression of estrogen receptors-α and -ß (ER-α and ER-ß) and similar antiviral effects were observed in the context of subgenomic DENV and ZIKV replicons, these compounds appear to inhibit viral RNA replication in a manner that is independent of their known effects on estrogen receptor signaling. Taken together, quinestrol, raloxifene hydrochloride, and structurally related analogues warrant further investigation as potential therapeutics for treatment of flavivirus infections.

Tenth Scientific Biennial Meeting of the Australasian Virology Society-AVS10 2019.

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2-5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on "Successfully transitioning from post-doc to lab head", winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.

A Recombinant Hepatitis C Virus Genotype 1a E1/E2 Envelope Glycoprotein Vaccine Elicits Antibodies That Differentially Neutralize Closely Related 2a Strains through Interactions of the N-Terminal Hypervariable Region 1 of E2 with Scavenger Receptor B1.

The global health burden for hepatitis C virus (HCV) remains high, despite available effective treatments. To eliminate HCV, a prophylactic vaccine is needed. One major challenge in the development of a vaccine is the genetic diversity of the virus, with 7 major genotypes and many subtypes. A global vaccine must be effective against all HCV genotypes. Our previous data showed that the 1a E1/E2 glycoprotein vaccine component elicits broad cross-neutralizing antibodies in humans and animals. However, some variation is seen in the effectiveness of these antibodies to neutralize different HCV genotypes and isolates. Of interest was the differences in neutralizing activity against two closely related isolates of HCV genotype 2a, the J6 and JFH-1 strains. Using site-directed mutagenesis to generate chimeric viruses between the J6 and JFH-1 strains, we found that variant amino acids within the core E2 glycoprotein domain of these two HCV genotype 2a viruses do not influence isolate-specific neutralization. Further analysis revealed that the N-terminal hypervariable region 1 (HVR1) of the E2 protein determines the sensitivity of isolate-specific neutralization, and the HVR1 of the resistant J6 strain binds scavenger receptor class-B type-1 (SR-B1), while the sensitive JFH-1 strain does not. Our data provide new information on mechanisms of isolate-specific neutralization to facilitate the optimization of a much-needed HCV vaccine.IMPORTANCE A vaccine is still urgently needed to overcome the hepatitis C virus (HCV) epidemic. It is estimated that 1.75 million new HCV infections occur each year, many of which will go undiagnosed and untreated. Untreated HCV can lead to continued spread of the disease, progressive liver fibrosis, cirrhosis, and eventually, end-stage liver disease and/or hepatocellular carcinoma (HCC). Previously, our 1a E1/E2 glycoprotein vaccine was shown to elicit broadly cross-neutralizing antibodies; however, there remains variation in the effectiveness of these antibodies against different HCV genotypes. In this study, we investigated determinants of differential neutralization sensitivity between two highly related genotype 2a isolates, J6 and JFH-1. Our data indicate that the HVR1 region determines neutralization sensitivity to vaccine antisera through modulation of sensitivity to antibodies and interactions with SR-B1. Our results provide additional insight into optimizing a broadly neutralizing HCV vaccine.