Publications by authors named "Rowena A Bull"

63 Publications

Optimisation and validation of a new method for antibody dependent cellular phagocytosis in hepatitis C virus infection.

J Immunol Methods 2021 Aug 18;495:113087. Epub 2021 Jun 18.

School of Medical Sciences, Faculty of Medicine, The University of New South Wales, Sydney, NSW 2052, Australia. Electronic address:

Lack of a simple, high throughput antibody-dependent cellular phagocytosis (ADCP) assay has limited our understanding of its potential role of in hepatitis C (HCV) infection. Here, we optimised a flow-cytometry based ADCP assay using HCV envelope (E2)-protein coated microbeads that were opsonised with anti-E2 monoclonal IgG antibody (αE2 mAb) and the THP-1 monocyte cell line as effector cells. We found 1.5 × 10/ml microbeads opsonised with 5 μg/ml αE2 mAb and 1.6 × 10/ml THP-1 cells were optimal conditions to distinguish between healthy controls and patients with HCV. This optimised assay was then used to investigate ADCP in plasma obtained from 72 patients with chronic HCV infection and 15 healthy controls. We found that 75% of patients with genotype 1 and 87% of patients with genotype 3 HCV infection had significantly higher levels of ADCP compared to healthy controls. In patients, there was a significant correlation between increase in ADCP and higher concentrations of anti-E2 IgG antibodies in the plasma. Taken together, we established a simple, quick and high throughput ADCP assay for HCV infection that can readily be used for screening of large cohorts of patients and investigation of the role of ADCP in the pathogenesis or protection from this disease.
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http://dx.doi.org/10.1016/j.jim.2021.113087DOI Listing
August 2021

Hepatitis C Virus Epitope Immunodominance and B Cell Repertoire Diversity.

Viruses 2021 May 25;13(6). Epub 2021 May 25.

Faculty of Medicine, School of Medical Sciences, UNSW Sydney, Sydney, NSW 2052, Australia.

Despite the advent of effective, curative treatments for hepatitis C virus (HCV), a preventative vaccine remains essential for the global elimination of HCV. It is now clear that the induction of broadly neutralising antibodies (bNAbs) is essential for the rational design of such a vaccine. This review details the current understanding of epitopes on the HCV envelope, characterising the potency, breadth and immunodominance of antibodies induced against these epitopes, as well as describing the interactions between B-cell receptors and HCV infection, with a particular focus on bNAb heavy and light chain variable gene usage. Additionally, we consider the importance of a public repertoire for antibodies against HCV, compiling current knowledge and suggesting that further research in this area may be critical to the rational design of an effective HCV vaccine.
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http://dx.doi.org/10.3390/v13060983DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8229270PMC
May 2021

Potent SARS-CoV-2 binding and neutralization through maturation of iconic SARS-CoV-1 antibodies.

MAbs 2021 Jan-Dec;13(1):1922134

Immunology Department, Garvan Institute of Medical Research, Sydney, NSW, Australia.

Antibodies against coronavirus spike protein potently protect against infection and disease, but whether such protection can be extended to variant coronaviruses is unclear. This is exemplified by a set of iconic and well-characterized monoclonal antibodies developed after the 2003 SARS outbreak, including mAbs m396, CR3022, CR3014 and 80R, which potently neutralize SARS-CoV-1, but not SARS-CoV-2. Here, we explore antibody engineering strategies to change and broaden their specificity, enabling nanomolar binding and potent neutralization of SARS-CoV-2. Intriguingly, while many of the matured clones maintained specificity of the parental antibody, new specificities were also observed, which was further confirmed by X-ray crystallography and cryo-electron microscopy, indicating that a limited set of VH antibody domains can give rise to variants targeting diverse epitopes, when paired with a diverse VL repertoire. Our findings open up over 15 years of antibody development efforts against SARS-CoV-1 to the SARS-CoV-2 field and outline general principles for the maturation of antibody specificity against emerging viruses.
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http://dx.doi.org/10.1080/19420862.2021.1922134DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158043PMC
May 2021

Human CD8 T-stem cell memory subsets phenotypic and functional characterization are defined by expression of CD122 or CXCR3.

Eur J Immunol 2021 Jul 23;51(7):1732-1747. Epub 2021 Apr 23.

Viral Immunology Systems Program (VISP), The Kirby Institute, University of New South Wales, Sydney, Australia.

Long-lived T-memory stem cells (T ) are key to both naturally occurring and vaccine-conferred protection against infection. These cells are characterized by the CD45RA CCR7 CD95 phenotype. Significant heterogeneity within the T population is recognized, but distinguishing surface markers and functional characterization of potential subsets are lacking. Human CD8 T subsets were identified in healthy subjects who had been previously exposed to CMV or Influenza (Flu) virus in flow cytometry by expression of CD122 or CXCR3, and then characterized in proliferation, multipotency, self-renewal, and intracellular cytokine production (TNF-α, IL-2, IFN-γ), together with transcriptomic profiles. The T CD122 -expressing subset (versus CD122 ) demonstrated greater proliferation, greater multipotency, and enhanced polyfunctionality with higher frequencies of triple positive (TNF-α, IL-2, IFN-γ) cytokine-producing cells upon exposure to recall antigen. The T CXCR3 subpopulation also had increased proliferation and polyfunctional cytokine production. Transcriptomic analysis further showed that the T CD122 population had increased expression of activation and homing molecules, such as Ccr6, Cxcr6, Il12rb, and Il18rap, and downregulated cell proliferation inhibitors, S100A8 and S100A9. These data reveal that the T CD122 phenotype is associated with increased proliferation, enhanced multipotency and polyfunctionality with an activated memory-cell like transcriptional profile, and hence, may be favored for induction by immunization and for adoptive immunotherapy.
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http://dx.doi.org/10.1002/eji.202049057DOI Listing
July 2021

Long-term persistence of RBD memory B cells encoding neutralizing antibodies in SARS-CoV-2 infection.

Cell Rep Med 2021 Apr 14;2(4):100228. Epub 2021 Mar 14.

The Kirby Institute, UNSW Australia, Sydney, NSW, Australia.

Considerable concerns relating to the duration of protective immunity against severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) exist, with evidence of antibody titers declining rapidly after infection and reports of reinfection. Here, we monitor the antibody responses against SARS-CoV-2 receptor-binding domain (RBD) for up to 6 months after infection. While antibody titers are maintained, ∼13% of the cohort's neutralizing responses return to background. However, encouragingly, in a selected subset of 13 participants, 12 have detectable RBD-specific memory B cells and these generally are increasing out to 6 months. Furthermore, we are able to generate monoclonal antibodies with SARS-CoV-2 neutralizing capacity from these memory B cells. Overall, our study suggests that the loss of neutralizing antibodies in plasma may be countered by the maintenance of neutralizing capacity in the memory B cell repertoire.
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http://dx.doi.org/10.1016/j.xcrm.2021.100228DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7955929PMC
April 2021

Respiratory viral co-infections among SARS-CoV-2 cases confirmed by virome capture sequencing.

Sci Rep 2021 02 16;11(1):3934. Epub 2021 Feb 16.

School of Women's and Children's Health, Faculty of Medicine, University of New South Wales, Sydney, NSW, Australia.

Accumulating evidence supports the high prevalence of co-infections among Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) patients, and their potential to worsen the clinical outcome of COVID-19. However, there are few data on Southern Hemisphere populations, and most studies to date have investigated a narrow spectrum of viruses using targeted qRT-PCR. Here we assessed respiratory viral co-infections among SARS-CoV-2 patients in Australia, through respiratory virome characterization. Nasopharyngeal swabs of 92 SARS-CoV-2-positive cases were sequenced using pan-viral hybrid-capture and the Twist Respiratory Virus Panel. In total, 8% of cases were co-infected, with rhinovirus (6%) or influenzavirus (2%). Twist capture also achieved near-complete sequencing (> 90% coverage, > tenfold depth) of the SARS-CoV-2 genome in 95% of specimens with Ct < 30. Our results highlight the importance of assessing all pathogens in symptomatic patients, and the dual-functionality of Twist hybrid-capture, for SARS-CoV-2 whole-genome sequencing without amplicon generation and the simultaneous identification of viral co-infections with ease.
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http://dx.doi.org/10.1038/s41598-021-83642-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7887263PMC
February 2021

SARS Coronavirus-2 Microneutralisation and Commercial Serological Assays Correlated Closely for Some but Not All Enzyme Immunoassays.

Viruses 2021 02 4;13(2). Epub 2021 Feb 4.

Virology Research Laboratory, Prince of Wales Hospital, Sydney, NSW 2031, Australia.

Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. Sera from recovered patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection ( = 200), and negative control sera collected prior to the COVID-19 pandemic ( = 100), were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. Neutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. These results suggest the marker used (total Ab vs. IgG vs. IgA) and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrates their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation.
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http://dx.doi.org/10.3390/v13020247DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7915197PMC
February 2021

Analytical validity of nanopore sequencing for rapid SARS-CoV-2 genome analysis.

Nat Commun 2020 12 9;11(1):6272. Epub 2020 Dec 9.

Kinghorn Centre for Clinical Genomics, Garvan Institute of Medical Research, Sydney, NSW, Australia.

Viral whole-genome sequencing (WGS) provides critical insight into the transmission and evolution of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2). Long-read sequencing devices from Oxford Nanopore Technologies (ONT) promise significant improvements in turnaround time, portability and cost, compared to established short-read sequencing platforms for viral WGS (e.g., Illumina). However, adoption of ONT sequencing for SARS-CoV-2 surveillance has been limited due to common concerns around sequencing accuracy. To address this, here we perform viral WGS with ONT and Illumina platforms on 157 matched SARS-CoV-2-positive patient specimens and synthetic RNA controls, enabling rigorous evaluation of analytical performance. We report that, despite the elevated error rates observed in ONT sequencing reads, highly accurate consensus-level sequence determination was achieved, with single nucleotide variants (SNVs) detected at >99% sensitivity and >99% precision above a minimum ~60-fold coverage depth, thereby ensuring suitability for SARS-CoV-2 genome analysis. ONT sequencing also identified a surprising diversity of structural variation within SARS-CoV-2 specimens that were supported by evidence from short-read sequencing on matched samples. However, ONT sequencing failed to accurately detect short indels and variants at low read-count frequencies. This systematic evaluation of analytical performance for SARS-CoV-2 WGS will facilitate widespread adoption of ONT sequencing within local, national and international COVID-19 public health initiatives.
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http://dx.doi.org/10.1038/s41467-020-20075-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7726558PMC
December 2020

Conserved epitopes with high HLA-I population coverage are targets of CD8 T cells associated with high IFN-γ responses against all dengue virus serotypes.

Sci Rep 2020 11 24;10(1):20497. Epub 2020 Nov 24.

Immunogenomics Laboratory, Kirby Institute for Infection and Immunity, University of New South Wales, Sydney, Australia.

Cytotoxic CD8 T cells are key for immune protection against viral infections. The breadth and cross-reactivity of these responses are important against rapidly mutating RNA viruses, such as dengue (DENV), yet how viral diversity affect T cell responses and their cross-reactivity against multiple variants of the virus remains poorly defined. In this study, an integrated analysis was performed to map experimentally validated CD8 T cell epitopes onto the distribution of DENV genome sequences across the 4 serotypes worldwide. Despite the higher viral diversity observed within HLA-I restricted epitopes, mapping of 609 experimentally validated epitopes sequences on 3985 full-length viral genomes revealed 19 highly conserved epitopes across the four serotypes within the immunogenic regions of NS3, NS4B and NS5. These conserved epitopes were associated with a higher magnitude of IFN-γ response when compared to non-conserved epitopes and were restricted to 13 HLA class I genotypes, hence providing high coverage among human populations. Phylogeographic analyses showed that these epitopes are largely conserved in most of the endemic regions of the world, and with only some of these epitopes presenting distinct mutated variants circulating in South America and Asia.This study provides evidence for the existence of highly immunogenic and conserved epitopes across serotypes, which may impact design of new universal T-cell-inducing vaccine candidates that minimise detrimental effects of viral diversification and at the same time induce responses to a broad human population.
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http://dx.doi.org/10.1038/s41598-020-77565-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7687909PMC
November 2020

Optimized cell systems for the investigation of hepatitis C virus E1E2 glycoproteins.

J Gen Virol 2021 01;102(1)

Institute of Immunity and Transplantation, Division of Infection and Immunity, The Royal Free Hospital, University College London, London, UK.

Great strides have been made in understanding and treating hepatitis C virus (HCV) thanks to the development of various experimental systems including cell-culture-proficient HCV, the HCV pseudoparticle system and soluble envelope glycoproteins. The HCV pseudoparticle (HCVpp) system is a platform used extensively in studies of cell entry, screening of novel entry inhibitors, assessing the phenotypes of clinically observed E1 and E2 glycoproteins and, most pertinently, in characterizing neutralizing antibody breadth induced upon vaccination and natural infection in patients. Nonetheless, some patient-derived clones produce pseudoparticles that are either non-infectious or exhibit infectivity too low for meaningful phenotyping. The mechanisms governing whether any particular clone produces infectious pseudoparticles are poorly understood. Here we show that endogenous expression of CD81, an HCV receptor and a cognate-binding partner of E2, in producer HEK 293T cells is detrimental to the infectivity of recovered HCVpp for most strains. Many HCVpp clones exhibited increased infectivity or had their infectivity rescued when they were produced in 293T cells CRISPR/Cas9 engineered to ablate CD81 expression (293T). Clones made in 293T cells were antigenically very similar to their matched counterparts made parental cells and appear to honour the accepted HCV entry pathway. Deletion of CD81 did not appreciably increase the recovered titres of soluble E2 (sE2). However, we did, unexpectedly, find that monomeric sE2 made in 293T cells and Freestyle 293-F (293-F) cells exhibit important differences. We found that 293-F-produced sE2 harbours mostly complex-type glycans whilst 293T-produced sE2 displays a heterogeneous mixture of both complex-type glycans and high-mannose or hybrid-type glycans. Moreover, sE2 produced in 293T cells is antigenically superior; exhibiting increased binding to conformational antibodies and the large extracellular loop of CD81. In summary, this work describes an optimal cell line for the production of HCVpp and reveals that sE2 made in 293T and 293-F cells are not antigenic equals. Our findings have implications for functional studies of E1E2 and the production of candidate immunogens.
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http://dx.doi.org/10.1099/jgv.0.001512DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8116788PMC
January 2021

Single molecule, near full-length genome sequencing of dengue virus.

Sci Rep 2020 10 23;10(1):18196. Epub 2020 Oct 23.

School of Medical Sciences, University of New South Wales, Sydney, Australia.

Current methods for dengue virus (DENV) genome amplification, amplify parts of the genome in at least 5 overlapping segments and then combine the output to characterize a full genome. This process is laborious, costly and requires at least 10 primers per serotype, thus increasing the likelihood of PCR bias. We introduce an assay to amplify near full-length dengue virus genomes as intact molecules, sequence these amplicons with third generation "nanopore" technology without fragmenting and use the sequence data to differentiate within-host viral variants with a bioinformatics tool (Nano-Q). The new assay successfully generated near full-length amplicons from DENV serotypes 1, 2 and 3 samples which were sequenced with nanopore technology. Consensus DENV sequences generated by nanopore sequencing had over 99.5% pairwise sequence similarity to Illumina generated counterparts provided the coverage was > 100 with both platforms. Maximum likelihood phylogenetic trees generated from nanopore consensus sequences were able to reproduce the exact trees made from Illumina sequencing with a conservative 99% bootstrapping threshold (after 1000 replicates and 10% burn-in). Pairwise genetic distances of within host variants identified from the Nano-Q tool were less than that of between host variants, thus enabling the phylogenetic segregation of variants from the same host.
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http://dx.doi.org/10.1038/s41598-020-75374-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7584602PMC
October 2020

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

Viruses 2020 06 6;12(6). Epub 2020 Jun 6.

Department of Microbiology and Immunology, The Peter Doherty Institute for Infection and Immunity, University of Melbourne, Melbourne, VIC 3000, Australia.

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.
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http://dx.doi.org/10.3390/v12060621DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7354434PMC
June 2020

Anti-envelope antibody responses in highly exposed seronegative individuals may be associated with protection from HCV infection.

J Viral Hepat 2020 10 17;27(10):1012-1021. Epub 2020 Jun 17.

Viral Immunology Systems Program, The Kirby Institute, The University of New South Wales, Sydney, New South Wales, Australia.

In rare cases, individuals with a history of long-term injecting drug use remain seronegative and aviraemic, despite prolonged and likely repeated exposure to Hepatitis C virus (HCV) through high-risk behaviour. We describe anti-HCV Envelope (E) antibody responses in a prospective cohort of carefully defined highly exposed but uninfected subjects (HESN) and comparison subjects who were also high risk and uninfected, but rapidly became HCV infected (Incident). Longitudinally collected samples from HESN cases (n = 22) were compared to Incident controls (n = 22). IgG, IgM and IgA from sera were tested by ELISA to genotype 1a and 3a E glycoproteins, and recombinant genotype 1a E2 antigen. IgG subclass isotyping was performed for those positive for IgG. Virus-neutralizing activity was assessed on HCV pseudoparticles, and HCV E-specific B cells analysed using flow cytometry. A significant minority of HESN cases (n = 10; 45%) had anti-E, predominantly in the IgG2 subclass, which was not found in the pre-infection time point of the Incident cases (n = 1; 5%). A subset of the HESN subjects also had neutralizing activity and HCV-specific B cells detected significantly more than Incident cases pre-infection. In conclusion, the HESN phenotype is associated with IgG2 anti-E antibodies, neutralization activity and HCV E-specific memory B cells. These findings suggest that HESN subjects may be resistant to HCV infection through humoral immune-mediated mechanisms.
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http://dx.doi.org/10.1111/jvh.13339DOI Listing
October 2020

Impact of an Open Access Nationwide Treatment Model on Hepatitis C Virus Antiviral Drug Resistance.

Hepatol Commun 2020 Jun 6;4(6):904-915. Epub 2020 Apr 6.

Storr Liver Centre The Westmead Institute for Medical Research The University of Sydney and Westmead Hospital Sydney Australia.

Direct acting antivirals (DAAs) have revolutionized hepatitis C virus (HCV) treatment, but drug resistance could undermine proposed global elimination targets. Real-world studies are needed to inform the impact of widespread DAA treatment on antiviral resistance in the community. The prevalence and range of posttreatment resistance-associated substitutions (RASs) was determined in Australian patients with open access to DAAs through a wide range of prescribers. NS3, NS5A, and NS5B regions were amplified by polymerase chain reaction and analyzed by population sequencing. Clinically relevant RASs were identified using online databases (ReCALL and Geno2Pheno[hcv]). Of 572 samples, 60% were from genotype 3 and 27% from genotype 1a. Ninety-two percent of people failed a DAA regimen containing an NS5A inhibitor, including 10% with a pangenotype regimen. NS5A RASs were detected in 72% of people with genotype 1 and 80% with genotype 3. For genotype 1, there was a range of RASs across the NS5A region, while for genotype 3, the Y93H RAS predominated (72%). The prevalence of NS3 RASs was higher in people exposed to an NS3 inhibitor (35% vs. 3.9%;  < 0.0001). NS5B resistance was rare, with a single case of sofosbuvir resistance. Multiclass drug resistance was found in 33% of people exposed to both NS3 and NS5A inhibitors. : The high prevalence of NS5A RASs among people failing DAA therapy reinforces the importance of specific retreatment regimens, ideally guided by resistance testing. The impact of multiclass drug resistance on retreatment in people exposed to both NS3 and NS5A inhibitors needs to be assessed in real-world studies. Surveillance for increasing antiviral resistance during treatment scale-up is essential to maintain the efficacy of current DAA regimens.
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http://dx.doi.org/10.1002/hep4.1496DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7262285PMC
June 2020

Envelope-Specific IgG3 and IgG1 Responses Are Associated with Clearance of Acute Hepatitis C Virus Infection.

Viruses 2020 01 8;12(1). Epub 2020 Jan 8.

Viral Immunology Systems Program, The Kirby Institute, Sydney, NSW 2052, Australia.

Hepatitis C virus (HCV) can be cleared naturally in a subset of individuals. However, the asymptomatic nature of acute HCV infection makes the study of the early immune response and defining the correlates of protection challenging. Despite this, there is now strong evidence implicating the humoral immune response, specifically neutralising antibodies, in determining the clearance or chronicity outcomes of primary HCV infection. In general, immunoglobulin G (IgG) plays the major role in viral neutralisation. However, there are limited investigations of anti-HCV envelope protein 2 (E2) isotypes (IgM, IgG, IgA) and IgG subclasses (IgG1-4) in early HCV infection. In this study, using a rare cohort of 14 very recently HCV-infected individuals (4-45 days) with varying disease outcome ( = 7 clearers), the timing and potency of anti-HCV E2 isotypes and IgG subclasses were examined longitudinally, in relation to neutralising antibody activity. Clearance was associated with anti-E2 IgG, specifically IgG1 and IgG3, and appeared essential to prevent the emergence of new HCV variants and the chronic infection outcome. Interestingly, these IgG responses were accompanied by IgM antibodies and were associated with neutralising antibody activity in the subjects who cleared infection. These findings provide novel insights into the early humoral immune response characteristics associated with HCV disease outcome.
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http://dx.doi.org/10.3390/v12010075DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7019651PMC
January 2020

B cell immunodominance in primary hepatitis C virus infection.

J Hepatol 2020 04 28;72(4):670-679. Epub 2019 Nov 28.

School of Medical Sciences and the Kirby Institute, Faculty of Medicine, UNSW Sydney, Sydney, NSW 2052, Australia. Electronic address:

Background & Aims: Neutralising antibodies (NAbs) play a key role in clearance of HCV. NAbs have been isolated and mapped to several domains on the HCV envelope proteins. However, the immunodominance of these epitopes in HCV infection remains unknown, hindering efforts to elicit optimal epitope-specific responses. Furthermore, it remains unclear which epitope-specific responses are associated with broad NAb (bNAb) activity in primary HCV infection. The aim of this study was to define B cell immunodominance in primary HCV, and its implications on neutralisation breadth and clearance.

Methods: Using samples from 168 patients with primary HCV infection, the antibody responses targeted 2 immunodominant domains, termed domains B and C. Genotype 1 and 3 infections were associated with responses targeted towards different bNAb domains.

Results: No epitopes were uniquely targeted by clearers compared to those who developed chronic infection. Samples with bNAb activity were enriched for multi-specific responses directed towards the epitopes antigenic region 3, antigenic region 4, and domain D, and did not target non-neutralising domains.

Conclusions: This study outlines for the first time a clear NAb immunodominance profile in primary HCV infection, and indicates that it is influenced by the infecting virus. It also highlights the need for a vaccination strategy to induce multi-specific responses that do not target non-neutralising domains.

Lay Summary: Neutralising antibodies will likely form a key component of a protective hepatitis C virus vaccine. In this work we characterise the predominant neutralising and non-neutralising antibody (epitope) targets in acute hepatitis C virus infection. We have defined the natural hierarchy of epitope immunodominance, and demonstrated that viral genotype can impact on this hierarchy. Our findings highlight key epitopes that are associated with broadly neutralising antibodies, and the deleterious impact of mounting a response towards some of these domains on neutralising breadth. These findings should guide future efforts to design immunogens aimed at generating neutralising antibodies with a vaccine candidate.
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http://dx.doi.org/10.1016/j.jhep.2019.11.011DOI Listing
April 2020

Clearance of hepatitis C virus is associated with early and potent but narrowly-directed, Envelope-specific antibodies.

Sci Rep 2019 09 16;9(1):13300. Epub 2019 Sep 16.

Viral Immunology Systems Program, The Kirby Institute, Sydney, Australia.

Hepatitis C virus (HCV) is one of very few viruses that are either naturally cleared, or alternatively persist to cause chronic disease. Viral diversity and escape, as well as host adaptive immune factors, are believed to control the outcome. To date, there is limited understanding of the critical, early host-pathogen interactions. The asymptomatic nature of early HCV infection generally prevents identification of the transmitted/founder (T/F) virus, and thus the study of host responses directed against the autologous T/F strain. In this study, 14 rare subjects identified from very early in infection (4-45 days) with varied disease outcomes (n = 7 clearers) were examined in regard to the timing, breadth, and magnitude of the neutralizing antibody (nAb) response, as well as evolution of the T/F strain. Clearance was associated with earlier onset and more potent nAb responses appearing at a mean of 71 days post-infection (DPI), but these responses were narrowly directed against the autologous T/F virus or closely related variants. In contrast, a delayed onset of nAbs (mean 425 DPI) was observed in chronic progressors that appear to have targeted longitudinal variants rather than the T/F strain. The nAb responses in the chronic progressors mapped to known CD81 binding epitopes, and were associated with rapid emergence of new viral variants with reduced CD81 binding. We propose that the prolonged period of viremia in the absence of nAbs in these subjects was associated with an increase in viral diversity, affording the virus greater options to escape nAb pressure once it emerged. These findings indicate that timing of the nAb response is essential for clearance. Further investigation of the specificities of the early nAbs and the factors regulating early induction of protective nAbs is needed.
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http://dx.doi.org/10.1038/s41598-019-49454-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6746763PMC
September 2019

A method for detecting hepatitis C envelope specific memory B cells from multiple genotypes using cocktail E2 tetramers.

J Immunol Methods 2019 09 19;472:65-74. Epub 2019 Jun 19.

School of Medical Sciences and the Kirby Institute, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia. Electronic address:

Hepatitis C (HCV) is a rapidly mutating RNA virus, with a strong propensity to cause chronic infection and progressive liver disease. Recent evidence has shown that early appearance of neutralizing antibodies in primary infection is associated with clearance. Little is known about the characteristics of HCV-specific B cells and their correlation with outcomes in primary infection, as there is a lack of sensitive tools for HCV-specific B cells which are present at very low frequency. We describe the development and optimisation of tetramer staining for flow cytometric detection of HCV-specific B cells using a cocktail of two recombinant HCV Envelope-2 (rE2) glycoproteins (from genotype 1a and 3a; Gt1a and Gt3a) and streptavidin dyes. The optimal weight to weight (w/w) ratio of streptavidin-phycoerythrin (PE) and rE2 proteins were determined for sensitive detection using HCV E2-specific hybridoma cell lines and peripheral blood mononuclear cells (PBMC) from HCV-infected individuals. In a cross-sectional set of PBMC samples collected from 33 subjects with either chronic infection or previous clearance, HCV E2-specific B cells (CD19CD20CD10IgDtetramer) were detected in 29 subjects (87.8%), with a mean frequency of 0.45% (0.012-2.20%). To validate the specificity of tetramer staining, 367 HCV E2-specific B cells were single cell sorted from 9 PBMC samples before monoclonal antibodies (mAbs) were synthesised, with 87.5% being reactive to E2 via ELISA. Of these mAbs, 284 and 246 clones were reactive to either Gt1a or Gt3a E2 proteins, respectively. This is a sensitive and robust method for future studies investigating B cell responses against the HCV Envelope protein.
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http://dx.doi.org/10.1016/j.jim.2019.06.016DOI Listing
September 2019

Broadly neutralizing antibodies from an individual that naturally cleared multiple hepatitis C virus infections uncover molecular determinants for E2 targeting and vaccine design.

PLoS Pathog 2019 05 17;15(5):e1007772. Epub 2019 May 17.

Department of Pathology, Stanford University School of Medicine, Stanford, California, United States of America.

Cumulative evidence supports a role for neutralizing antibodies contributing to spontaneous viral clearance during acute hepatitis C virus (HCV) infection. Information on the timing and specificity of the B cell response associated with clearance is crucial to inform vaccine design. From an individual who cleared three sequential HCV infections with genotypes 1b, 1a and 3a strains, respectively, we employed peripheral B cells to isolate and characterize neutralizing human monoclonal antibodies (HMAbs) to HCV after the genotype 1 infections. The majority of isolated antibodies, designated as HMAbs 212, target conformational epitopes on the envelope glycoprotein E2 and bound broadly to genotype 1-6 E1E2 proteins. Further, some of these antibodies showed neutralization potential against cultured genotype 1-6 viruses. Competition studies with defined broadly neutralizing HCV HMAbs to epitopes in distinct clusters, designated antigenic domains B, C, D and E, revealed that the selected HMAbs compete with B, C and D HMAbs, previously isolated from subjects with chronic HCV infections. Epitope mapping studies revealed domain B and C specificity of these HMAbs 212. Sequential serum samples from the studied subject inhibited the binding of HMAbs 212 to autologous E2 and blocked a representative domain D HMAb. The specificity of this antibody response appears similar to that observed during chronic infection, suggesting that the timing and affinity maturation of the antibody response are the critical determinants in successful and repeated viral clearance. While additional studies should be performed for individuals with clearance or persistence of HCV, our results define epitope determinants for antibody E2 targeting with important implications for the development of a B cell vaccine.
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http://dx.doi.org/10.1371/journal.ppat.1007772DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6542541PMC
May 2019

Toward DNA-Based T-Cell Mediated Vaccines to Target HIV-1 and Hepatitis C Virus: Approaches to Elicit Localized Immunity for Protection.

Front Cell Infect Microbiol 2019 3;9:91. Epub 2019 Apr 3.

Virology Laboratory, Basil Hetzel Institute for Translational Health Research, Discipline of Surgery, University of Adelaide, Adelaide, SA, Australia.

Human immunodeficiency virus (HIV)-1 and hepatitis C virus (HCV) are major contributors to the global disease burden with many experts recognizing the requirement of an effective vaccine to bring a durable end to these viral epidemics. The most promising vaccine candidates that have advanced into pre-clinical models and the clinic to eliminate or provide protection against these chronic viruses are viral vectors [e.g., recombinant cytomegalovirus, Adenovirus, and modified vaccinia Ankara (MVA)]. This raises the question, is there a need to develop DNA vaccines against HIV-1 and HCV? Since the initial study from Wolff and colleagues which showed that DNA represents a vector that can be used to express transgenes durably , DNA has been regularly evaluated as a vaccine vector albeit with limited success in large animal models and humans. However, several recent studies in Phase I-IIb trials showed that vaccination of patients with recombinant DNA represents a feasible therapeutic intervention to even cure cervical cancer, highlighting the potential of using DNA for human vaccinations. In this review, we will discuss the limitations and the strategies of using DNA as a vector to develop prophylactic T cell-mediated vaccines against HIV-1 and HCV. In particular, we focus on potential strategies exploiting DNA vectors to elicit protective localized CD8 T cell immunity in the liver for HCV and in the cervicovaginal mucosa for HIV-1 as localized immunity will be an important, if not critical component, of an efficacious vaccine against these viral infections.
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http://dx.doi.org/10.3389/fcimb.2019.00091DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6456646PMC
December 2019

Genomic characterization of hepatitis C virus transmitted founder variants with deep sequencing.

Infect Genet Evol 2019 07 8;71:36-41. Epub 2019 Mar 8.

School of Medical Sciences, Faculty of Medicine, UNSW, Sydney, NSW, Australia. Electronic address:

Transfer of hepatitis C virus (HCV) infection from a donor to a new recipient is associated with a bottleneck of genetic diversity in the transmitted viral variants. Existing data suggests that one, or very few, variants emerge from this bottleneck to establish the infection (transmitted founder [T/F] variants). In HCV, very few T/F variants have been characterized due to the challenges of obtaining early infection samples and of high throughput viral genome sequencing. This study used a large, acute HCV, deep-sequenced dataset from first viremia samples collected in nine prospective cohorts across four countries, to estimate the prevalence of single T/F viruses, and to identify host and virus-related factors associated with infections initiated by a single T/F variant. The short reads generated by Illumina sequencing were used to reconstruct viral haplotypes with two haplotype reconstruction algorithms. The haplotypes were examined for random mutations (Poisson distribution) and a star-like phylogeny to identify T/F viruses. The findings were cross-validated by haplotype reconstructions across three regions of the genome (Core-E2, NS3, NS5A) to minimize the possibility of spurious overestimation of single T/F variants. Of 190 acute infection samples examined, 54 were very early acute infections (HCV antibody negative, RNA positive), and single transmitted founders were identified in 14 (26%, 95% CI: 16-39%) after cross validation across multiple regions of the genome with two haplotype reconstruction algorithms. The presence of a single T/F virus was not associated with any host or virus-related factors, notably viral genotype or spontaneous clearance. In conclusion, approximately one in four new HCV infections originates from a single T/F virus. Resolution of genomic sequences of single T/F variants is the first step in exploring unique properties of these variants in the infection of host hepatocytes.
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http://dx.doi.org/10.1016/j.meegid.2019.02.032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6487228PMC
July 2019

Higher abundance of enterovirus A species in the gut of children with islet autoimmunity.

Sci Rep 2019 02 11;9(1):1749. Epub 2019 Feb 11.

School of Women's and Children's Health, University of New South Wales Faculty of Medicine, Sydney, Australia.

Enteroviruses (EVs) are prime candidate environmental triggers of islet autoimmunity (IA), with potential as vaccine targets for type 1 diabetes prevention. However, the use of targeted virus detection methods and the selective focus on EVs by most studies increases the risk for substantial investigation bias and an overestimated association between EV and type 1 diabetes. Here we performed comprehensive virome-capture sequencing to examine all known vertebrate-infecting viruses without bias in 182 specimens (faeces and plasma) collected before or at seroconversion from 45 case children with IA and 48 matched controls. From >2.6 billion reads, 28 genera of viruses were detected and 62% of children (58/93) were positive for ≥1 vertebrate-infecting virus. We identified 129 viruses as differentially abundant between the gut of cases and controls, including 5 EV-A types significantly more abundant in the cases. Our findings further support EV's hypothesised contribution to IA and corroborate the proposal that viral load may be an important parameter in disease pathogenesis. Furthermore, our data indicate a previously unrecognised association of IA with higher EV-A abundance in the gut of children and provide a catalog of viruses to be interrogated further to determine a causal link between virus infection and type 1 diabetes.
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http://dx.doi.org/10.1038/s41598-018-38368-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370883PMC
February 2019

Genomic variability of within-host hepatitis C variants in acute infection.

J Viral Hepat 2019 04 22;26(4):476-484. Epub 2019 Jan 22.

School of Medical Sciences, UNSW, Sydney, New South Wales, Australia.

Interactions between the host immune system and the viral variants determine persistence of hepatitis C virus (HCV) infection after the acute phase of infection. This study describes the genetic variability of within-host HCV viral variants in acute infection and correlates it with host- and virus-related traits and infection outcome. Next generation sequence data (Illumina, MiSeq platform) of viral genomes from 116 incident acute infections (within 180 days of infection) were analysed to determine all the single nucleotide polymorphism (SNP) frequencies above a threshold of 0.1%. The variability of the SNPs for the full open reading frame of the genome as well as for each protein coding region were compared using mean standardized Shannon entropy (SE) values calculated separately for synonymous and nonsynonymous mutations. The envelope glycoproteins regions (E1 and E2) had the highest SE values (indicating greater variability) followed by the NS5B region. Nonsynonymous mutations rather than synonymous mutations were the main contributors to genomic variability in acute infection. The mean difference of Shannon entropy was also compared between subjects after categorizing the samples according to host and virus-related traits. Host IFNL3 allele CC polymorphism at rs12979860 (vs others) and viral genotype 1a (vs 3a) were associated with higher genomic variability across the viral open reading frame. Time since infection, host gender or continent of origin was not associated with the viral genomic variability. Viral genomic variability did not predict spontaneous clearance.
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http://dx.doi.org/10.1111/jvh.13051DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6417964PMC
April 2019

Chemokine-Regulated Recruitment of Antigen-Specific T-Cell Subpopulations to the Liver in Acute and Chronic Hepatitis C Infection.

J Infect Dis 2019 04;219(9):1430-1438

Viral Immunology Systems Program, The Kirby Institute.

Background: In hepatitis C virus (HCV) infection, virus-specific CD8+ T cells are recruited to the liver for antiviral activity. Multiple chemokine ligands are induced by the infection, notably interferon-inducible chemokine, CXCL10. In HCV, intrahepatic T cells express chemokine receptors (CCRs), including CXCR3, CXCR6, CCR1, and CCR5, but CCR expression on antigen-specific effector and memory T cells has not been investigated.

Methods: Paired blood and liver samples were collected from subjects with chronic HCV for flow cytometric analysis of CCR expression on CD8+ T cells. Expression of these CCRs was then examined on HCV-specific CD8+ T-cell subpopulations in the blood from subjects with acute or chronic HCV.

Results: Relative to peripheral blood, the liver was enriched with CD8+ T cells expressing CCR2, CCR5, CXCR3, and CXCR6 either singly or in combinations. CXCR3 was preferentially expressed on HCV-specific CD8+ T cells in both acute and chronic phases of infection in blood. Both CXCR3 and CCR2 were overexpressed on HCV-specific CD8+CCR7+CD45RO+ (central memory) cells, whereas effector memory (CD8+CCR7-CD45RO+) cells expressed more CXCR6.

Conclusions: CXCR3-mediated signals support the accumulation of HCV-specific CD8+ memory T cells in the infected liver, and emphasize the importance of the CXCL10/CXCR3 trafficking pathway during acute and chronic HCV infection.
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http://dx.doi.org/10.1093/infdis/jiy679DOI Listing
April 2019

Understanding the Determinants of BnAb Induction in Acute HCV Infection.

Viruses 2018 11 21;10(11). Epub 2018 Nov 21.

School of Medical Sciences and the Kirby Institute, Faculty of Medicine, UNSW Australia, Sydney, NSW 2052, Australia.

Despite recent advances in curative therapy, hepatitis C virus (HCV) still remains a global threat. In order to achieve global elimination, a prophylactic vaccine should be considered high priority. Previous immunogens used to induce broad neutralising antibodies (BnAbs) have been met with limited success. To improve immunogen design, factors associated with the early development of BnAbs in natural infection must first be understood. In this study, 43 subjects identified with acute HCV were analysed longitudinally using a panel of heterogeneous HCV pseudoparticles (HCVpp), to understand the emergence of BnAbs. Compared to those infected with a single genotype, early BnAb development was associated with subjects co-infected with at least 2 HCV subtypes during acute infection. In those that were mono-infected, BnAbs were seen to emerge with increasing viral persistence. If subjects acquired a secondary infection, nAb breadth was seen to boost upon viral re-exposure. Importantly, this data highlights the potential for multivalent and prime-boost vaccine strategies to induce BnAbs against HCV in humans. However, the data also indicate that the infecting genotype may influence the development of BnAbs. Therefore, the choice of antigen will need to be carefully considered in future vaccine trials.
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http://dx.doi.org/10.3390/v10110659DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6266478PMC
November 2018

Amplification and next generation sequencing of near full-length human enteroviruses for identification and characterisation from clinical samples.

Sci Rep 2018 08 8;8(1):11889. Epub 2018 Aug 8.

School of Women's and Children's Health, Faculty of Medicine, University of New South Wales, Sydney, NSW, 2052, Australia.

More than 100 different enterovirus (EV) genotypes infect humans and contribute to substantial morbidity. However, current methods for characterisation of full-length genomes are based on Sanger sequencing of short genomic regions, which are labour-intensive and do not enable comprehensive characterisation of viral populations. Here, we describe a simple and sensitive protocol for the amplification and sequencing of near full-length genomes of human EV species using next generation sequencing. EV genomes were amplified from 89% of samples tested, with C values ranging between 15.7 and 39.3. These samples included 7 EV-A genotypes (CVA2, 5-7, 10, 16 and EV71), 19 EV-B genotypes (CVA9, CVB1-6, ECHO3, 4, 6, 7, 9, 11, 16, 18, 25, 29, 30, and EV69), 3 EV-C genotypes (CVA19 and PV2, 3) and 1 EV-D genotype (EV70). We characterised 70 EVs from 58 clinical stool samples and eight reference strains, with a minimum of 100X depth. We found evidence of co-infection in four clinical specimens, each containing two distinct EV genotypes (CVB3/ECHO7, CVB3/ECHO18 and ECHO9/30). Characterisation of the complete genome provided conclusive genotyping of EVs, which can be applied to investigate the intra-host virus evolution of EVs, and allows further identification and investigation of EV outbreaks.
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http://dx.doi.org/10.1038/s41598-018-30322-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6082906PMC
August 2018

Clonally diverse CD38HLA-DRCD8 T cells persist during fatal H7N9 disease.

Nat Commun 2018 02 26;9(1):824. Epub 2018 Feb 26.

Shanghai Public Health Clinical Center & Institutes of Biomedical Sciences, Key Laboratory of Medical Molecular Virology of Ministry of Education/Health, Shanghai Medical College, Fudan University, 201508, Shangai, China.

Severe influenza A virus (IAV) infection is associated with immune dysfunction. Here, we show circulating CD8 T-cell profiles from patients hospitalized with avian H7N9, seasonal IAV, and influenza vaccinees. Patient survival reflects an early, transient prevalence of highly activated CD38HLA-DRPD-1 CD8 T cells, whereas the prolonged persistence of this set is found in ultimately fatal cases. Single-cell T cell receptor (TCR)-αβ analyses of activated CD38HLA-DRCD8 T cells show similar TCRαβ diversity but differential clonal expansion kinetics in surviving and fatal H7N9 patients. Delayed clonal expansion associated with an early dichotomy at a transcriptome level (as detected by single-cell RNAseq) is found in CD38HLA-DRCD8 T cells from patients who succumbed to the disease, suggesting a divergent differentiation pathway of CD38HLA-DRCD8 T cells from the outset during fatal disease. Our study proposes that effective expansion of cross-reactive influenza-specific TCRαβ clonotypes with appropriate transcriptome signatures is needed for early protection against severe influenza disease.
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http://dx.doi.org/10.1038/s41467-018-03243-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5827521PMC
February 2018

Sequencing of hepatitis C virus for detection of resistance to direct-acting antiviral therapy: A systematic review.

Hepatol Commun 2017 07 22;1(5):379-390. Epub 2017 May 22.

Kirby Institute University of New South Wales Sydney Australia.

The significance of the clinical impact of direct-acting antiviral (DAA) resistance-associated substitutions (RASs) in hepatitis C virus (HCV) on treatment failure is unclear. No standardized methods or guidelines for detection of DAA RASs in HCV exist. To facilitate further evaluations of the impact of DAA RASs in HCV, we conducted a systematic review of RAS sequencing protocols, compiled a comprehensive public library of sequencing primers, and provided expert guidance on the most appropriate methods to screen and identify RASs. The development of standardized RAS sequencing protocols is complicated due to a high genetic variability and the need for genotype- and subtype-specific protocols for multiple regions. We have identified several limitations of the available methods and have highlighted areas requiring further research and development. The development, validation, and sharing of standardized methods for all genotypes and subtypes should be a priority. ( 2017;1:379-390).
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http://dx.doi.org/10.1002/hep4.1050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5721421PMC
July 2017

Genome-Wide Mutagenesis of Dengue Virus Reveals Plasticity of the NS1 Protein and Enables Generation of Infectious Tagged Reporter Viruses.

J Virol 2017 12 14;91(23). Epub 2017 Nov 14.

Research Centre for Infectious Diseases, School of Biological Sciences, University of Adelaide, Adelaide, Australia

Dengue virus (DENV) is a major global pathogen that causes significant morbidity and mortality in tropical and subtropical areas worldwide. An improved understanding of the regions within the DENV genome and its encoded proteins that are required for the virus replication cycle will expedite the development of urgently required therapeutics and vaccines. We subjected an infectious DENV genome to unbiased insertional mutagenesis and used next-generation sequencing to identify sites that tolerate 15-nucleotide insertions during the virus replication cycle in hepatic cell culture. This revealed that the regions within capsid, NS1, and the 3' untranslated region were the most tolerant of insertions. In contrast, prM- and NS2A-encoding regions were largely intolerant of insertions. Notably, the multifunctional NS1 protein readily tolerated insertions in regions within the , , and β- domains with minimal effects on viral RNA replication and infectious virus production. Using this information, we generated infectious reporter viruses, including a variant encoding the APEX2 electron microscopy tag in NS1 that uniquely enabled high-resolution imaging of its localization to the surface and interior of viral replication vesicles. In addition, we generated a tagged virus bearing an mScarlet fluorescent protein insertion in NS1 that, despite an impact on fitness, enabled live cell imaging of NS1 localization and traffic in infected cells. Overall, this genome-wide profile of DENV genome flexibility may be further dissected and exploited in reporter virus generation and antiviral strategies. Regions of genetic flexibility in viral genomes can be exploited in the generation of reporter virus tools and should arguably be avoided in antiviral drug and vaccine design. Here, we subjected the DENV genome to high-throughput insertional mutagenesis to identify regions of genetic flexibility and enable tagged reporter virus generation. In particular, the viral NS1 protein displayed remarkable tolerance of small insertions. This genetic flexibility enabled generation of several novel NS1-tagged reporter viruses, including an APEX2-tagged virus that we used in high-resolution imaging of NS1 localization in infected cells by electron microscopy. For the first time, this analysis revealed the localization of NS1 within viral replication factories known as "vesicle packets" (VPs), in addition to its acknowledged localization to the luminal surface of these VPs. Together, this genetic profile of DENV may be further refined and exploited in the identification of antiviral targets and the generation of reporter virus tools.
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http://dx.doi.org/10.1128/JVI.01455-17DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5686748PMC
December 2017

Persistent infections in immunocompromised hosts are rarely sources of new pathogen variants.

Virus Evol 2017 Jul 28;3(2):vex018. Epub 2017 Jul 28.

School of Biotechnology and Biomolecular Sciences, and Evolution & Ecology Research Centre, University of New South Wales, Sydney, NSW 2052, Australia.

Many viruses, including human norovirus and influenza, cause self-limiting diseases of short duration. However, infection by the same viruses in an immunocompromised host can result in prolonged illness in the absence of effective treatment. Such persistent infections are often characterized by increased genetic diversity with potentially elevated rates of evolution compared to acute infections, leading to suggestions that immunocompromised hosts represent an important reservoir for the emergence of novel viral variants. Here, we develop a mathematical model that combines epidemiological dynamics with within-host evolution to quantify the relative contribution of immunocompromised hosts to the overall rate of pathogen evolution. Using human norovirus as a case study we show that the majority of evolutionary substitutions are expected to occur in acute infections of immunocompetent hosts. Hence, despite their potential to generate a high level of diversity, infections of immunocompromised hosts likely contribute less to the evolution and emergence of new genetic variants at the epidemiological scale because such hosts are rare and tend to be isolated. This result is robust to variation in key parameters, including the proportion of the population immunocompromised, and provides a means to understand the adaptive significance of mutations that arise during chronic infections in immunocompromised hosts.
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http://dx.doi.org/10.1093/ve/vex018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5534129PMC
July 2017
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