Publications by authors named "Narayan Ramamurthy"

24 Publications

  • Page 1 of 1

Molecular characterization of porcine circovirus 2 circulating in Assam and Arunachal Pradesh of India.

Anim Biotechnol 2021 Aug 10:1-5. Epub 2021 Aug 10.

Recombinant DNA Laboratory, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Bareilly, India.

PCV2 is the primary etiological agent of porcine circovirus-associated diseases (PCVADs) which affect pigs worldwide. Currently, there is a worldwide genotype prevalence switch from PCV2b to PCV2d, which has led to increased virulence of the circulating virus strains leading to vaccine failures and selection pressure. In the present study, the PCV2 genotypes circulating in north eastern region (NER) of India particularly the states of Assam and Arunachal Pradesh was characterized by isolation, sequencing and phylogenetic analysis of gene. The phylogenetic analysis revealed that the PCV2 isolates circulating in pigs of Assam and Arunachal Pradesh were mostly of PCV2d genotype. Hence, it can be concluded that PCV2d genotype is the most dominating genotype in NER and priority should be given to this genotype for development of future vaccine candidate against PCV2 in India.
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http://dx.doi.org/10.1080/10495398.2021.1955700DOI Listing
August 2021

Evaluation of the oncolytic property of recombinant Newcastle disease virus strain R2B in 4T1 and B16-F10 cells in-vitro.

Res Vet Sci 2021 Oct 25;139:159-165. Epub 2021 Jul 25.

Recombinant DNA Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India. Electronic address:

Recombinant Newcastle disease virus vectors have gained a lot of interest for its oncolytic virus therapy and cancer immune therapeutic properties due to its selective replication to high titers in cancer cells. The aim of this study was to find out the oncolytic effects of mesogenic recombinant NDV strain R2B-GFP on murine mammary tumor cell line 4T1 and murine melanoma cell line B16-F10. The anti-tumor effects of R2B-GFP virus were studied via expression of virus transgene GFP in cancer cells, evaluating its cytotoxicity and cell migration efficacies by MTT and wound healing assays respectively. In addition, the underlying apoptotic mechanism of R2B-GFP virus was estimated by TUNEL assay, colorimetric estimation of Caspase-3, 8 and 9 and the estimation of Bax to Bcl-2 ratio. The results showed a significant decrease in viability of both 4T1 and B16-F10 cells infected with R2B-GFP virus at 0.1 and 1 MOI. R2B-GFP virus could significantly induce apoptosis in the 4T1 and B16-F10 cells as compared to the uninfected control. Further, a flow cytometry analysis on apoptotic cells percentage and mitochondria membrane permeability test was also studied in R2B-GFP virus treated 4T1 and B16-F10 cell lines. The R2B-GFP virus caused an increase in loss of mitochondrial membrane permeability in both 4T1 and B16-F10 cells indicating the involvement of mitochondrial regulated cell death. Thus, the recombinant virus R2B-GFP virus proved to be a valid candidate for oncolytic viral therapy in 4T1 and B16-F10 cells.
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http://dx.doi.org/10.1016/j.rvsc.2021.07.028DOI Listing
October 2021

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses.

Nat Commun 2021 04 6;12(1):2055. Epub 2021 Apr 6.

Jenner Institute, University of Oxford, Oxford, UK.

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.
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http://dx.doi.org/10.1038/s41467-021-21856-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8024333PMC
April 2021

Detection of neutralising antibodies to SARS-CoV-2 to determine population exposure in Scottish blood donors between March and May 2020.

Euro Surveill 2020 10;25(42)

The members of the ISARIC4C Investigators are listed under the investigator tab.

BackgroundThe progression and geographical distribution of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection in the United Kingdom (UK) and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of blood donors in Scotland in the spring of 2020 to detect neutralising antibodies to SARS-CoV-2 as a marker of past infection and epidemic progression.AimOur objective was to determine if sera from blood bank donors can be used to track the emergence and progression of the SARS-CoV-2 epidemic.MethodsA pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study comprised samples from 3,500 blood donors collected in Scotland between 17 March and 18 May 2020. Controls were collected from 100 donors in Scotland during 2019.ResultsAll samples collected on 17 March 2020 (n = 500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in six of 500 donors from 23 to 26 March. The number of samples containing neutralising antibodies did not significantly rise after 5-6 April until the end of the study on 18 May. We found that infections were concentrated in certain postcodes, indicating that outbreaks of infection were extremely localised. In contrast, other areas remained comparatively untouched by the epidemic.ConclusionAlthough blood donors are not representative of the overall population, we demonstrated that serosurveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic such as the SARS-CoV-2 outbreak.
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http://dx.doi.org/10.2807/1560-7917.ES.2020.25.42.2000685DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7651873PMC
October 2020

Newcastle disease virus vectored rabies vaccine induces strong humoral and cell mediated immune responses in mice.

Vet Microbiol 2020 Dec 12;251:108890. Epub 2020 Oct 12.

Recombinant DNA Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly, 243 122, India. Electronic address:

Rabies is a devastating disease affecting almost all mammalian animal species including humans. Vaccines are available to combat the disease. Protection against the disease is rendered by assessing the humoral immune response. Recent reports suggest the role of cell mediated immune response (CMI) in assessing vaccine efficacy. In the present study, two live vectored vaccine candidates containing glycoprotein G of rabies virus were generated using the mesogenic Newcastle disease virus (NDV) strain R2B and another with NDV with an altered fusion protein cleavage site as backbones. The efficacy of these vaccine candidates on testing in experimental mouse model indicated generation of robust humoral and CMI responses. The recombinant NDV containing the altered fusion protein cleavage site with glycoprotein G showed the highest CMI response in mice indicating its usage as a potential live vectored vaccine candidate against the disease.
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http://dx.doi.org/10.1016/j.vetmic.2020.108890DOI Listing
December 2020

Systemic iron reduction by venesection alters the gut microbiome in patients with haemochromatosis.

JHEP Rep 2020 Dec 28;2(6):100154. Epub 2020 Jul 28.

Translational Gastroenterology Unit, University of Oxford, Oxford, UK.

Background & Aims: Iron reduction by venesection has been the cornerstone of treatment for haemochromatosis for decades, and its reported health benefits are many. Repeated phlebotomy can lead to a compensatory increase in intestinal iron absorption, reducing intestinal iron availability. Given that most gut bacteria are highly dependent on iron for survival, we postulated that, by reducing gut iron levels, venesection could alter the gut microbiota.

Methods: Clinical parameters, faecal bacterial composition and metabolomes were assessed before and during treatment in a group of patients with haemochromatosis undergoing iron reduction therapy.

Results: Systemic iron reduction was associated with an alteration of the gut microbiome, with changes evident in those who experienced reduced faecal iron availability with venesection. For example, levels of , a bacterium associated with improved colonic health, were increased in response to faecal iron reduction. Similarly, metabolomic changes were seen in association with reduced faecal iron levels.

Conclusion: These findings highlight a significant shift in the gut microbiome of patients who experience reduced colonic iron during venesection. Targeted depletion of faecal iron could represent a novel therapy for metabolic and inflammatory diseases, meriting further investigation.

Lay Summary: Iron depletion by repeated venesection is the mainstay of treatment for haemochromatosis, an iron-overload disorder. Venesection has been associated with several health benefits, including improvements in liver function tests, reversal of liver scarring, and reduced risk of liver cancer. During iron depletion, iron absorption from the gastrointestinal (GI) tract increases to compensate for iron lost with treatment. Iron availability is limited in the GI tract and is crucial to the growth and function of many gut bacteria. In this study we show that reduced iron availability in the colon following venesection treatment leads to a change in the composition of the gut bacteria, a finding that, to date, has not been studied in patients with haemochromatosis.
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http://dx.doi.org/10.1016/j.jhepr.2020.100154DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7516344PMC
December 2020

Recombinant Newcastle Disease Virus (NDV) Expressing Sigma C Protein of Avian Reovirus (ARV) Protects against Both ARV and NDV in Chickens.

Pathogens 2019 Sep 10;8(3). Epub 2019 Sep 10.

Recombinant DNA Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243 122, India.

Newcastle disease (ND) and avian reovirus (ARV) infections are a serious threat to the poultry industry, which causes heavy economic losses. The mesogenic NDV strain R2B is commonly used as a booster vaccine in many Asian countries to control the disease. In this seminal work, a recombinant NDV strain R2B expressing the sigma C (σC) gene of ARV (rNDV-R2B-σC) was generated by reverse genetics, characterized in vitro and tested as a bivalent vaccine candidate in chickens. The recombinant rNDV-R2B-σC virus was attenuated as compared to the parent rNDV-R2B virus as revealed by standard pathogenicity assays. The generated vaccine candidate, rNDV-R2B-σC, could induce both humoral and cell mediated immune responses in birds and gave complete protection against virulent NDV and ARV challenges. Post-challenge virus shedding analysis revealed a drastic reduction in NDV shed, as compared to unvaccinated birds.
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http://dx.doi.org/10.3390/pathogens8030145DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6789743PMC
September 2019

Infectious bursal disease virus in chickens: prevalence, impact, and management strategies.

Vet Med (Auckl) 2019 5;10:85-97. Epub 2019 Aug 5.

Recombinant DNA Lab, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly 243122, India.

Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of young chickens. Although first observed about 60 years ago, to date, the disease is responsible for major economic losses in the poultry industry worldwide. IBD virus (IBDV), a double-stranded RNA virus, exists as two serotypes with only serotype 1 causing the disease in young chickens. The virus infects the bursa of Fabricius of particularly the actively dividing and differentiating lymphocytes of the B-cells lineage of immature chickens, resulting in morbidity, mortality, and immunosuppression. Immunosuppression enhances the susceptibility of chickens to other infections and interferes with vaccination against other diseases. Immunization is the most important measure to control IBD; however, rampant usage of live vaccines has resulted in the evolution of new strains. Although the immunosuppression caused by IBDV is more directed toward the B lymphocytes, the protective immunity in birds depends on inducement of both humoral and cell-mediated immune responses. The interference with the inactivated vaccine induced maternally derived antibodies in young chicks has become a hurdle in controlling the disease, thus necessitating the development of newer vaccines with improved efficacy. The present review illustrates the overall dynamics of the virus and the disease, and the recent developments in the field of virus diagnosis and vaccine research.
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http://dx.doi.org/10.2147/VMRR.S185159DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6689097PMC
August 2019

Evaluation of a fusion gene-based DNA prime-protein boost vaccination strategy against Newcastle disease virus.

Trop Anim Health Prod 2019 Nov 18;51(8):2529-2538. Epub 2019 Jun 18.

Recombinant DNA Laboratory, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly, UP, 243122, India.

The low potency of genetic immunization has to date impeded development of commercial vaccines against major infectious diseases. The aim of this study was to develop and evaluate a fusion gene-based DNA prime-protein boost vaccination strategy to improve the efficacy of both DNA and subunit vaccines against Newcastle disease virus (NDV). The fusion (F) protein, a viral surface glycoprotein, is responsible for the cell membrane fusion and spread, also is one of the major targets for immune response. In this study, groups of chickens were vaccinated twice intramuscularly at 14-day interval either with plasmid DNA encoding F protein gene of NDV or with recombinant F protein alone or with plasmid DNA and boosted with the recombinant F protein and compared with birds that were vaccinated with live NDV vaccine. The immune response was evaluated by indirect ELISA, lymphocyte transformation test, virus neutralization test, cytokine analysis, immunophenotyping of peripheral blood mononuclear cells, and protective efficacy study against virulent NDV challenge virus infection. Chickens in prime-boost group developed a higher level of humoral and cellular immune responses as compared with those immunized with plasmid or protein alone. The DNA prime-protein boost using F protein of NDV yielded 91.6% protection against virulent NDV challenge infection better than immunization with DNA vaccine (66.6%) or rF protein (83.3%) alone. These findings suggest that the "DNA prime-protein boost" approach using full-length F gene could enhance the immune response against NDV in the chickens.
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http://dx.doi.org/10.1007/s11250-019-01967-2DOI Listing
November 2019

Serological profiling of rabies antibodies by enzyme-linked immunosorbent assay and its comparative analysis with rapid fluorescent focus inhibition test in mouse model.

Vet World 2019 Jan 23;12(1):126-130. Epub 2019 Jan 23.

Recombinant DNA Laboratory, Division of Veterinary Biotechnology, Indian Veterinary Research Institute, Izatnagar, Bareilly, Uttar Pradesh, India.

Aim: In this study, we have used enzyme-linked immunosorbent assay (ELISA) as an alternative test to replace the cumbersome rapid fluorescent focus inhibition test (RFFIT) to ascertain the immune status of immunized mice against rabies virus.

Materials And Methods: Rabies is a devastating disease worldwide caused by rabies virus. Proper usage of pre- or post-exposure rabies vaccine can prevent the disease transmission. In this study, mice were immunized with Vero cell-adapted inactivated rabies vaccine. RFFIT was used as a test to determine the serum neutralizing titers in infected/vaccinated mice. Seroprofiling of mice sera was done by ELISA.

Results: Twenty-one days post-immunization, both ELISA and RFFIT assays indicated similar antibody levels in mice sera that were immunized with Vero cell-adapted inactivated rabies vaccine. Both the tests were correlated, and the linearity was verified by the regression line (R²=0.979).

Conclusion: In this study, we profiled the serological status of Vero cell-adapted inactivated rabies vaccine through ELISA in mice model that correlated well with the OIE gold standard test RFFIT.
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http://dx.doi.org/10.14202/vetworld.2019.126-130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6431817PMC
January 2019

Antiviral activity of bone morphogenetic proteins and activins.

Nat Microbiol 2019 02 3;4(2):339-351. Epub 2018 Dec 3.

MRC Human Immunology Unit, MRC Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, UK.

Understanding the control of viral infections is of broad importance. Chronic hepatitis C virus (HCV) infection causes decreased expression of the iron hormone hepcidin, which is regulated by hepatic bone morphogenetic protein (BMP)/SMAD signalling. We found that HCV infection and the BMP/SMAD pathway are mutually antagonistic. HCV blunted induction of hepcidin expression by BMP6, probably via tumour necrosis factor (TNF)-mediated downregulation of the BMP co-receptor haemojuvelin. In HCV-infected patients, disruption of the BMP6/hepcidin axis and genetic variation associated with the BMP/SMAD pathway predicted the outcome of infection, suggesting that BMP/SMAD activity influences antiviral immunity. Correspondingly, BMP6 regulated a gene repertoire reminiscent of type I interferon (IFN) signalling, including upregulating interferon regulatory factors (IRFs) and downregulating an inhibitor of IFN signalling, USP18. Moreover, in BMP-stimulated cells, SMAD1 occupied loci across the genome, similar to those bound by IRF1 in IFN-stimulated cells. Functionally, BMP6 enhanced the transcriptional and antiviral response to IFN, but BMP6 and related activin proteins also potently blocked HCV replication independently of IFN. Furthermore, BMP6 and activin A suppressed growth of HBV in cell culture, and activin A inhibited Zika virus replication alone and in combination with IFN. The data establish an unappreciated important role for BMPs and activins in cellular antiviral immunity, which acts independently of, and modulates, IFN.
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http://dx.doi.org/10.1038/s41564-018-0301-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6590058PMC
February 2019

Impact of Interferon Lambda 4 Genotype on Interferon-Stimulated Gene Expression During Direct-Acting Antiviral Therapy for Hepatitis C.

Hepatology 2018 09;68(3):859-871

Peter Medawar Building for Pathogen Research and Translational Gastroeneterology Unit, Nuffield Department of Medicine, University of Oxford, Oxford, United Kingdom.

New directly acting antivirals (DAAs) provide very high cure rates in most patients infected by hepatitis C virus (HCV). However, some patient groups have been relatively harder to treat, including those with cirrhosis or infected with HCV genotype 3. In the recent BOSON trial, genotype 3, patients with cirrhosis receiving a 16-week course of sofosbuvir and ribavirin had a sustained virological response (SVR) rate of around 50%. In patients with cirrhosis, interferon lambda 4 (IFNL4) CC genotype was significantly associated with SVR. This genotype was also associated with a lower interferon-stimulated gene (ISG) signature in peripheral blood and in liver at baseline. Unexpectedly, patients with the CC genotype showed a dynamic increase in ISG expression between weeks 4 and 16 of DAA therapy, whereas the reverse was true for non-CC patients. Conclusion: These data provide an important dynamic link between host genotype and phenotype in HCV therapy also potentially relevant to naturally acquired infection. (Hepatology 2018; 00:000-000).
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http://dx.doi.org/10.1002/hep.29877DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6207923PMC
September 2018

Role of Cell-Penetrating Peptides in Intracellular Delivery of Peptide Nucleic Acids Targeting Hepadnaviral Replication.

Mol Ther Nucleic Acids 2017 Dec 14;9:162-169. Epub 2017 Sep 14.

INSERM U1052, Cancer Research Center of Lyon (CRCL), University of Lyon, Lyon, France. Electronic address:

Peptide nucleic acids (PNAs) are potentially attractive antisense agents against hepatitis B virus (HBV), although poor cellular uptake limits their therapeutic application. In the duck HBV (DHBV) model, we evaluated different cell-penetrating peptides (CPPs) for delivery to hepatocytes of a PNA-targeting hepadnaviral encapsidation signal (ε). This anti-ε PNA exhibited sequence-specific inhibition of DHBV RT in a cell-free system. Investigation of the best in vivo route of delivery of PNA conjugated to (D-Arg) (P1) showed that intraperitoneal injection to ducklings was ineffective, whereas intravenously (i.v.) injected fluorescein-P1-PNA reached the hepatocytes. Treatment of virus carriers with i.v.-administered P1-PNA resulted in a decrease in viral DNA compared to untreated controls. Surprisingly, a similar inhibition of viral replication was observed in vivo as well as in vitro in primary hepatocyte cultures for a control 2 nt mismatched PNA conjugated to P1. By contrast, the same PNA coupled to (D-Lys) (P2) inhibited DHBV replication in a sequence-specific manner. Interestingly, only P1, but not P2, displayed anti-DHBV activity in the absence of PNA cargo. Hence, we provide new evidence that CPP-PNA conjugates inhibit DHBV replication following low-dose administration. Importantly, our results demonstrate the key role of CPPs used as vehicles in antiviral specificity of CPP-PNA conjugates.
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http://dx.doi.org/10.1016/j.omtn.2017.09.003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5633256PMC
December 2017

Impact of IL-27 on hepatocyte antiviral gene expression and function.

Wellcome Open Res 2016 Nov 17;1:17. Epub 2016 Nov 17.

Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK.

Interleukin (IL)-27 is a member of the IL-6/IL-12 family of cytokines. It is a potent cytokine, with potential antiviral impact, and has been shown to play a role in modulating functions of diverse cell types, including Th1, Th2, and NK and B cells, demonstrating both pro- and anti-inflammatory roles.  In hepatocytes, it is capable of inducing signal transducer and activator of transcription (STAT)1, STAT3 and interferon-stimulated genes. To address its role in viral hepatitis, the antiviral activity of IL-27 against hepatitis C virus (HCV) and hepatitis B virus (HBV) was tested using cell-culture-derived infectious HCV (HCVcc) cell culture system and the HepaRG HBV cell culture model. To further investigate the impact of IL-27 on hepatocytes, Huh7.5 cells were treated with IL-27 to analyse the differentially expressed genes by microarray analysis. Furthermore, by quantitative PCR, we analyzed the up-regulation of chemokine in response to IL-27. In both HCV and HBV infection models, we observed only a modest direct antiviral effect. Microarray analysis showed that the up-regulated genes mostly belonged to antigen presentation and DNA replication pathways, and involved strong up-regulation of , a gene associated with liver inflammation. Overall, gene set enrichment analysis showed a striking correlation of these genes with those up-regulated in response to related cytokines in diverse cell populations. Our data indicate that IL-27 can have a significant pro-inflammatory impact , although the direct antiviral effect is modest. It may have a potential impact on hepatocyte function, especially chemokine expression and antigen presentation.
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http://dx.doi.org/10.12688/wellcomeopenres.9917.1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5207303PMC
November 2016

MAIT cells are activated during human viral infections.

Nat Commun 2016 06 23;7:11653. Epub 2016 Jun 23.

Peter Medawar Building for Pathogen Research and Translational Gastroenterology Unit, Nuffield Department of Clinical Medicine, University of Oxford, Oxford OX1 3SY, UK.

Mucosal-associated invariant T (MAIT) cells are abundant in humans and recognize bacterial ligands. Here, we demonstrate that MAIT cells are also activated during human viral infections in vivo. MAIT cells activation was observed during infection with dengue virus, hepatitis C virus and influenza virus. This activation-driving cytokine release and Granzyme B upregulation-is TCR-independent but dependent on IL-18 in synergy with IL-12, IL-15 and/or interferon-α/β. IL-18 levels and MAIT cell activation correlate with disease severity in acute dengue infection. Furthermore, HCV treatment with interferon-α leads to specific MAIT cell activation in vivo in parallel with an enhanced therapeutic response. Moreover, TCR-independent activation of MAIT cells leads to a reduction of HCV replication in vitro mediated by IFN-γ. Together these data demonstrate MAIT cells are activated following viral infections, and suggest a potential role in both host defence and immunopathology.
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http://dx.doi.org/10.1038/ncomms11653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4931007PMC
June 2016

Liver sinusoidal endothelial cells: an antiviral "defendothelium".

Gastroenterology 2015 Feb 18;148(2):288-91. Epub 2014 Dec 18.

NIHR Biomedical Research Centre and Nuffield Department of Medicine, Peter Medawar Building for Pathogen Research, University of Oxford, Oxford, UK.

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http://dx.doi.org/10.1053/j.gastro.2014.12.010DOI Listing
February 2015

Human MAIT and CD8αα cells develop from a pool of type-17 precommitted CD8+ T cells.

Blood 2012 Jan 15;119(2):422-33. Epub 2011 Nov 15.

Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, United Kingdom.

Human mucosal associated invariant T (MAIT) CD8(+) and Tc17 cells are important tissue-homing cell populations, characterized by high expression of CD161 ((++)) and type-17 differentiation, but their origins and relationships remain poorly defined. By transcriptional and functional analyses, we demonstrate that a pool of polyclonal, precommitted type-17 CD161(++)CD8αβ(+) T cells exist in cord blood, from which a prominent MAIT cell (TCR Vα7.2(+)) population emerges post-natally. During this expansion, CD8αα T cells appear exclusively within a CD161(++)CD8(+)/MAIT subset, sharing cytokine production, chemokine-receptor expression, TCR-usage, and transcriptional profiles with their CD161(++)CD8αβ(+) counterparts. Our data demonstrate the origin and differentiation pathway of MAIT-cells from a naive type-17 precommitted CD161(++)CD8(+) T-cell pool and the distinct phenotype and function of CD8αα cells in man.
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http://dx.doi.org/10.1182/blood-2011-05-353789DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3257008PMC
January 2012

Molecular footprints reveal the impact of the protective HLA-A*03 allele in hepatitis C virus infection.

Gut 2011 Nov 6;60(11):1563-71. Epub 2011 May 6.

Department of Clinical Medicine, Trinity College Dublin, St James' Hospital, Dublin, Ireland.

Background And Aims: CD8 T cells are central to the control of hepatitis C virus (HCV) although the key features of a successful CD8 T cell response remain to be defined. In a cohort of Irish women infected by a single source, a strong association between viral clearance and the human lecucocyte (HLA)-A*03 allele has been described, and the aim of this study was to define the protective nature of the associated CD8 T cell response.

Methods: A sequence-led approach was used to identify HLA-A*03-restricted epitopes. We examine the CD8 T cell response associated with this gene and address the likely mechanism underpinning this protective effect in this special cohort, using viral sequencing, T cell assays and analysis of fitness of viral mutants.

Results: A strong 'HLA footprint' in a novel NS3 epitope (TVYHGAGTK) was observed. A lysine (K) to arginine (R) substitution at position 9 (K1088R) was seen in a significant number of A*03-positive patients (9/12) compared with the control group (1/33, p=0.0003). Threonine (T) was also substituted with alanine (A) at position 8 (T1087A) more frequently in A*03-positive patients (6/12) compared with controls (2/33, p=0.01), and the double substitution of TK to AR was also observed predominantly in HLA-A*03-positive patients (p=0.004). Epitope-specific CD8 T cell responses were observed in 60% of patients three decades after exposure and the mutants selected in vivo impacted on recognition in vitro. Using HCV replicons matched to the viral sequences, viral fitness was found to be markedly reduced by the K1088R substitution but restored by the second substitution T1087A.

Conclusions: It is proposed that at least part of the protective effect of HLA-A*03 results from targeting of this key epitope in a functional site: the requirement for two mutations to balance fitness and escape provides an initial host advantage. This study highlights the potential protective impact of common HLA-A alleles against persistent viruses, with important implications for HCV vaccine studies.
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http://dx.doi.org/10.1136/gut.2010.228403DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184218PMC
November 2011

Transcriptome sequencing, microarray, and proteomic analyses reveal cellular and metabolic impact of hepatitis C virus infection in vitro.

Hepatology 2010 Aug;52(2):443-53

Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, UK.

Unlabelled: Hepatitis C virus (HCV) is a major cause of liver disease but the full impact of HCV infection on the hepatocyte is poorly understood. RNA sequencing (RNA-Seq) is a novel method to analyze the full transcriptional activity of a cell or tissue, thus allowing new insight into the impact of HCV infection. We conducted the first full-genome RNA-Seq analysis in a host cell to analyze infected and noninfected cells, and compared this to microarray and proteomic analyses. The combined power of the triple approach revealed that HCV infection affects a number of previously unreported canonical pathways and biological functions, including pregnane X receptor/retinoic acid receptor activation as a potential host antiviral response, and integrin-linked kinase signaling as an entry factor. This approach also identified several mechanisms implicated in HCV pathogenesis, including an increase in reactive oxygen species. HCV infection had a broad effect on cellular metabolism, leading to increases in cellular cholesterol and free fatty acid levels, associated with a profound and specific decrease in cellular glucose levels.

Conclusion: RNA-Seq technology, especially when combined with established methods, demonstrated that HCV infection has potentially wide-ranging effects on cellular gene and protein expression. This in vitro study indicates a substantial metabolic impact of HCV infection and highlights new mechanisms of virus-host interaction which may be highly relevant to pathogenesis in vivo.
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http://dx.doi.org/10.1002/hep.23733DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3427885PMC
August 2010

Analysis of CD161 expression on human CD8+ T cells defines a distinct functional subset with tissue-homing properties.

Proc Natl Acad Sci U S A 2010 Feb 28;107(7):3006-11. Epub 2010 Jan 28.

Department of Medicine II and Spemann Graduate School of Biology and Medicine, University of Freiburg, 79106 Freiburg, Germany.

CD8(+) T lymphocytes play a key role in host defense, in particular against important persistent viruses, although the critical functional properties of such cells in tissue are not fully defined. We have previously observed that CD8(+) T cells specific for tissue-localized viruses such as hepatitis C virus express high levels of the C-type lectin CD161. To explore the significance of this, we examined CD8(+)CD161(+) T cells in healthy donors and those with hepatitis C virus and defined a population of CD8(+) T cells with distinct homing and functional properties. These cells express high levels of CD161 and a pattern of molecules consistent with type 17 differentiation, including cytokines (e.g., IL-17, IL-22), transcription factors (e.g., retinoic acid-related orphan receptor gamma-t, P = 6 x 10(-9); RUNX2, P = 0.004), cytokine receptors (e.g., IL-23R, P = 2 x 10(-7); IL-18 receptor, P = 4 x 10(-6)), and chemokine receptors (e.g., CCR6, P = 3 x 10(-8); CXCR6, P = 3 x 10(-7); CCR2, P = 4 x 10(-7)). CD161(+)CD8(+) T cells were markedly enriched in tissue samples and coexpressed IL-17 with high levels of IFN-gamma and/or IL-22. The levels of polyfunctional cells in tissue was most marked in those with mild disease (P = 0.0006). These data define a T cell lineage that is present already in cord blood and represents as many as one in six circulating CD8(+) T cells in normal humans and a substantial fraction of tissue-infiltrating CD8(+) T cells in chronic inflammation. Such cells play a role in the pathogenesis of chronic hepatitis and arthritis and potentially in other infectious and inflammatory diseases of man.
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http://dx.doi.org/10.1073/pnas.0914839107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2840308PMC
February 2010

Proteomic analysis of HepaRG cells: a novel cell line that supports hepatitis B virus infection.

J Proteome Res 2009 Jan;8(1):118-22

Oxford Antiviral Drug Discovery Unit, Oxford, OX1 3QU, UK.

The first proteomic characterization of the HepaRG cell line, the only cell line that is susceptible to hepatitis B virus (HBV) infection and supports a complete virus life cycle, is reported. Differential analysis of naive and HBV-infected HepaRG cells by two-dimensional gel electrophoresis revealed 19 differentially regulated features, 7 increasing and 12 decreasing with HBV infection. The proteins identified in these features were involved in various cellular pathways including apoptosis, DNA/RNA processing, and hepatocellular impairment. Similar expression changes in a number of the identified proteins have already been reported for other virus systems. Identification of these expression changes is a validation of the proteomics approach and contributes to an understanding of host cellular response to HBV infection.
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http://dx.doi.org/10.1021/pr800562jDOI Listing
January 2009

Rise in gamma interferon expression during resolution of duck hepatitis B virus infection.

J Gen Virol 2006 Nov;87(Pt 11):3225-3232

INSERM Unit 271, 151 Cours Albert Thomas, 69003 Lyon, France.

Gamma interferon (IFN-gamma) expression plays a crucial role in the control of mammalian hepatitis B virus (HBV) infection. However, the role of duck INF-gamma (DuIFN-gamma) in the outcome of duck HBV (DHBV) infection, a reference model for hepadnavirus replication studies, has not yet been investigated. This work explored the dynamics of DuIFN-gamma expression in liver and peripheral blood mononuclear cells (PBMCs) during resolution of DHBV infection in adolescent ducks in relation to serum and liver markers of virus replication, histological changes and humoral response induction. DHBV infection of 3-week-old ducks resulted in transient expression of intrahepatic preS protein (days 3-14) and mild histological changes. Low-level viraemia was detected only during the first 10 days of infection and was accompanied by early anti-preS antibody response induction. Importantly, a strong increase in intrahepatic DuIFN-gamma RNA was detected by real-time RT-PCR at days 6-14, which coincided with a sharp decrease in both viral DNA and preS protein in the liver. Interestingly, liver DuIFN-gamma expression remained augmented to the end of the follow-up period (day 66) and correlated with portal lymphocyte infiltration and persistence of trace quantities of intrahepatic DHBV DNA in animals that had apparently completely resolved the infection. Moreover, in infected ducks, a moderate increase was detected in the levels of DuIFN-gamma in PBMCs (days 12-14), which coincided with the peak in liver DuIFN-gamma RNA levels. These data reveal that increased DuIFN-gamma expression in liver and PBMCs is concomitant with viral clearance, characterizing the resolution of infection, and provide new insights into the host-virus interactions that control DHBV infection.
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http://dx.doi.org/10.1099/vir.0.82170-0DOI Listing
November 2006

Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA).

J Hepatol 2005 Feb;42(2):180-7

INSERM U271, 151 Cours Albert Thomas, F-69424 Lyon Cedex 03, France.

Background/aims: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT) and to compare their efficacy with phosphorothioate oligodeoxynucleotides (S-ODNs).

Methods: The effect of two partly overlapping PNAs targeting epsilon and of analogous S-ODNs was tested in cell-free transcription and translation system for DHBV RT expression. In addition their antiviral effect was investigated in primary duck hepatocytes (PDH).

Results: Both PNAs reproducibly inhibited DHBV RT in a dose-dependent manner with IC(50) of 10nM, whereas up to 600-fold higher concentration of S-ODNs was required for similar inhibition. The PNA targeting the bulge and upper stem of epsilon appeared as more efficient RT inhibitor than the PNA targeting only the bulge. Importantly, the inhibition was highly sequence-specific since double-mismatched PNA had no effect on the RT reaction. Moreover, in PDH the PNA coupled to Arg(7) cationic delivery peptide decreased DHBV replication.

Conclusions: We provide the first evidence that PNAs targeting the bulge and upper stem of epsilon can efficiently and in a sequence-specific manner inhibit DHBV RT.
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http://dx.doi.org/10.1016/j.jhep.2004.10.010DOI Listing
February 2005
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