ESTABLISHED AND EMERGING BLOOD-BORNE VIRUSES Juraj Petrik, PhD Head, Microbiology R&D, Scottish National Blood Transfusion Service & Honorary Senior Lecturer, University of Edinburgh Bratislava, 12 March 2008 OUTLINE Human pathogens Blood borne viruses (BBV) Established and emerging BBV: characterization, epidemiology, pathogenicity, treatment Highly pathogenic global BBV Highly pathogenic endemic BBV BBV pathogenic for certain patient groups Pathogen detection, blood screening and residual risk of transfusion Burden of BBV infections: global individual Human pathogens 1, 407 → 58 % zoonotic; 177 emerging or reemerging Most zoonotic pathogens not transmissible or minimally transmissible between humans. 25 % some person-to-person transmission. Number Considered emerging Bacteria: 538 54 (10 %) Fungi: 317 22 (7 %) Protozoa: 57 14 (25 %) Helminths: 287 10 (3 %) Viruses: 208 77 (37 %) More than 20 families, but Bunyaviridae, Flaviviridae, Togaviridae, Reoviridae accounting for > 50 % [MEJ Woolhouse, S Gowtage-Sequeria, Emerging Infection Diseases (Dec 2005)11:1842] Reasons for accelerated appearance of infectious diseases 1 Urbanisation → high density population areas → poor social & hygienic conditions 2 Increasing international travel (~ 2.1 billion airline passengers in 2006) 3 Increased migration: economy, result of conflict, forced displacement 4 Increasing international trade → unintentional spread of pathogens, vectors.. 5 Changes in climate and habitat 6 Iatrogenic spread of unknown pathogens 7 Emergence of mutants, resistant strains to available drugs 8 Changes in intervention policies (e.g. widespread insecticide use) 9 Vaccination coverage (detrimental effect of conflicts, lack of funding, unsubstantiated rumours etc.) Blood-borne viruses (BBV) BBV: can be transmitted via blood, blood products, body fluids, cells, tissues, organs. Established and emerging BBV: Established: well understood pathogenesis, transmission; available diagnostic tests; routinely tested for (at least on a proportion of donations). Emerging: limited knowledge; regional, if any, routine testing. Highly pathogenic global BBV: HBV HIV HCV [HTLV I, II] Highly pathogenic endemic BBV: WNV → WNV Dengue Chikungunya [HAV, HEV] BBV pathogenic for certain patient groups: CMV B19 → B19 LCMV [HHV 6,8] [Enterovirus ?] BBV with non- established disease association: [Anelloviruses, HGV] HBV - hepatitis B virus; HIV - human immunodeficiency virus; HCV - hepatitis C virus; HTLV - human T-cell lymphotropic virus; WNV - West Nile virus; HAV, HEV - Hepatitis A, E virus; CMV - Cytomegalovirus; B19 - Parvovirus B19; LCMV - Lymphocytic choriomeningitis virus; HHV 6, 8 - Human herpesvirus 6, 8 Highly pathogenic global BBV HBV, HIV, HCV, [HTLV I, II] Typical blood-borne viruses Blood - related transmission: main transmission route Often life-long infection High morbidity and mortality High global burden of infections worldwide Routinely screened for in blood donations Low residual risk of transfusion Hepatitis B virus (HBV): Virion and Genome Virion: ø42 nm; enveloped (Dane particle) Genome: partially dS DNA (- strand complete); 3.2 kb; 5’cohesive ends 4 overlapping orfs coding for: Surface S Core C Polymerase P (DNA poly, RT, RNase H) Transactivating X Genotypes: A-F Replication: 1 2 3 4 DNA repaired to fully complementary , closed circle 4 RNAs synthesized 3.5 kb RNA reverse transcribed One DNA species for DNA amplification, another for virion HBV: Transmission, Prevalence, Infection Transmission: vertical - most prevalent worldwide transfusion and other medical procedures intravenous drug use sexual Prevalence: One of the most prevalent viruses 350 - 400 million chronic carriers worldwide Sub-Saharan Africa, most of Asia, Western Pacific Amazon, Central, Eastern, Southern Europe India, Middle East Australia, N.Zealand, Northern & Western Europe, Northern America Infection: 8 – 10 % chronic inf. 2–7% 5% <2 % 10 % children & 30-50 % adults: symptomatic 2 - 10 % chronic infection 15 – 20 % cirrhosis/Hepatocellular carcinoma (HCC) 1 million deaths worldwide (acute & chronic hepatitis, HCC) HBV: Infection (cont.) and Pathogenesis Infection - continued HBsAg used as a general marker of infection anti-HBc IgM marker of acute infection anti-HBcIgG past or chronic infection HBeAg indicates active replication of virus and therefore infectiveness. Anti-HBe virus no longer replicating. However, the patient can still be positive for HBsAg which is made by integrated HBV. HBV-DNA indicates active replication of the virus, more accurate than HBeAg especially in cases of escape mutants. Used mainly for monitoring response to therapy. Pathogenesis: Noncythopatic virus. Most liver damage through immune, especially CTL responses, with contribution from antigen-nonspecific inflammatory cells Occult hepatitis B (after resolved acute hepatitis B, or asymptomatic HBV exposure): Presence of HBV DNA in: serum cells of the immune system and/or hepatic tissue Absence of serum HBsAg Accumulating data due to the NAT detecting low levels of HBV DNA HBV: Prevention and Treatment Vaccine: Available since 1982- subunit; later recombinant (yeast) 95 % effective in preventing chronic infection development 116 countries - routine immunisation programme Routine vaccination of 0-18 year olds Vaccination of risk groups of all ages Treatment: six drugs used for the treatment of persons with chronic hepatitis B: Adefovir dipivoxil interferon alfa-2b pegylated interferon alfa-2a lamivudine entecavir telbivudine Human immunodeficiency virus (HIV): Virion and genome Lentivirus, Retroviridae Virion: 100 - 120 nm; enveloped Genome: 2 copies of non-covalently linked 9.4 kb RNA+ Genotypes: HIV-1: M (99.6 %), N, O M A, B, C, D, F, G, K - subtypes E, I - CFR (circulating recombinant forms) predominant: C: 47.2 %) A and CRF02_AG: 27 % B: 12.3 % HIV-2: subtypes A - G; A (0.11 %) and B most prevalent Proteins: gag, pol, env regulatory: vif, vpr, tat, rev, nef, Vpx(HIV-2), Vpu (HIV-1) HIV: Transmission and prevalence Transmission: unprotected sex contaminated needles breast milk from an infected mother to her baby at birth (20 -30 %) blood, blood products - almost eliminated in developed countries Prevalence: WHO 2007 estimates: 33.2 million people living with HIV over 60 % in sub-Saharan Africa ~ 15 % South East Asia 2.5 million new infections 2.1 million deaths HIV: Infection and pathogenesis Infection: CD4 CCR5 (almost all individuals initially infected with CCR5-trophic virus; 32 aminoacid deletion homozygots protected) CXCR4 Mannose-specific C-type lectin receptors such as DC-SIGN (dendritic cells) Huge reservoir of replicating virus (1010 infected cells in an average patient) Pathogenesis: Slow destruction of T-cells, in particular central memory cells, necessary for lifelong protection against viruses 1 direct viral killing of infected cells 2 increased rates of apoptosis in infected cells 3 killing of infected CD4+ T cells by CD8 cytotoxic lymphocytes that recognize infected cells HIV: Prevention and Treatment Preventative vaccine remains elusive 23 approved drugs (USA, 2007) 8 NRTI (nucleoside or nucleotide reverse transcriptase inhibitors): Zidovudine, Didanosine, Zalcitabine, Stavudine, lamivudine, Abacavir, Tenofovir, Emtricitabine 3 NNRTI (non-nucleoside reverse transcriptase inhibitors): Nevirapine, Efavirenz, Delavirdine 10 PI (protease inhibitors): Saquinavir, Indinavir, Rotonavir, Nelfinavir, Amprenavir, Lopinavir + Ritonavir, Atazanavir, Fosamprenavir, Tripanavir, Darunavir 2 E/F I (entry or fusion inhibitors): Enfuvirtide, Maraviroc Need for new drugs: resistant mutants better tolerability lower toxicity co-formulations: better treatment adherence Hepatitis C virus (HCV): Virus Hepacivirus; Flaviviridae Virion: Enveloped, 55 - 65 nm Genome: 9.6 kb sS RNA + Coding for polyprotein, subsequently cleaved: Genotypes: structural: non-structural: C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, NS5B 1 -6 (> 100 subtypes) Replication: RNA poly error rate: 1 in 10,000 to 1 in 100,000 After: www.med.uni-heidelberg.de HCV: Transmission and prevalence Transmission: injectable drug use blood, blood products vertical (3 - 15 % risk) Prevalence: ~ 180 million people infected worldwide: USA 3 - 4 million Europe 15 million Asia 90 - 95 million Africa 30 - 40 million Americas 12 -15 million It is estimated only about ½ diagnosed in developed countries <1% Australia, Canada, Northern Europe ~1% USA, most of Europe >2% Africa, Latin America, Central & South-Eastern Asia, (some countries 5 - 10 %) 19 % (10 - 19 years old persons) - 60 % (30 years old persons) - Nile delta Egypt HCV: Infection and pathogenesis Infection: CD81, SR-BI, [LDL rec, L/DC-SIGN] 60 - 70 % % asymptomatic ~50 - 80 % becomes chronic Pathogenesis: 10 -20 % of chronically-infected individuals develop cirrhosis, 1 - 5 % HCC HCV responsible for : 27 % cirrhosis 50 - 76 % of all liver cancers (25 % HCC) 2/3 of all liver transplants Mechanism: both, the immune-system-mediated pathobiological changes and direct viral cytopathic effects HCV: Prevention and Treatment Prevention: no vaccine In the absence of a vaccine, all precautions to prevent infection must be taken including: Screening and testing of blood and organ donors; Virus inactivation of plasma derived products; Implementation and maintenance of infection control practices in health care settings, including appropriate sterilization of medical and dental equipment; Promotion of behaviour change among the general public and health care workers to reduce overuse of injections and to use safe injection practices; Risk reduction counselling for persons with high-risk drug and sexual practices. Treatment: pegylated interferon and ribavirin: success rate depends on genotype: 1: up to 50 % 2&3: 50 - 80 % Highly pathogenic endemic viruses Mostly emerging: WNV (→ WNV), Dengue, Chikungunya, [HAV, HEV] Defined primarily as ‘arthropode-borne’ or mosquito-borne’ (WNV, Dengue, Chikungunya) or ‘ food or water-borne’ (HAV, HEV) But: Emerging bloodborne pathogens include new and re-emerging agents that can be transmitted through blood, blood products, body fluids, and biological therapeutic products, including cells, tissues, and organs Increasing territorial spread, following spread of suitable vectors, probably as a consequence of the climate change Significant morbidity and mortality High global burden of infections West Nile Virus (WNV) Flavivirus, Flaviviridae Virion: ~50 nm, enveloped Genome: sS RNA -; 11kb; coding for: structural C, E, M and seven NS proteins Lineages 1 and 2 (recently circulating in Central Europe) Infection: Majority asymptomatic Minority: Mild (fever) Neuroinvasive - ~ 10 % deaths In infected individuals the ratio between the three states is roughly 110:30:1. Host range: Mostly birds Susceptible mammalian species: horse, dog, man Transmission: Mosquito - Culex and 16 other species Blood, blood products Prevalence: Africa, Asia (India, Indonesia), Australia, Southern Europe, Eastern Europe (co-circulation of 1 and 2) 1999 introduction to USA (lineage 1) WNV US EPIDEMICS 1999 2002 2005 2000 2003 2006 2001 2004 2007 WNV: Statistics of US epidemics Year Overall clinical cases Viremic blood donors Fatalities 1999 62 nd 7 2000 21 nd 2 2001 66 nd 9 2002 4156 nd 284 2003 9861 818 264 2004 2539 224 100 2005 3000 417 119 2006 4269 361 177 2007 3576 332 115 nd not determined Figures from CDC Dengue virus Flavivirus; Flaviviridae Virion: 50 nm, enveloped Genome: sS RNA - 4 serotypes DENV 1 - 4 Transmission: most common mosquito-borne viral disease Aedes aegypti (urban species); Aedes albopictus Prevalence: 2.5 billion people at risk of infection >100 countries: Africa, Americas, Eastern Mediterranean, South-east Asia, Western Pacific Dengue virus - continued Infection: Mosquito bite; 2 - 7 day viraemia Pathogenesis: ~50 - 100 million / year Dengue fever (DF) ~ 1% dengue hemorragic fever (DHF) and Dengue shock syndrome (DSS) ~ 2.5 % mortality (up to 20 % without treatment; ~ 1 % with support treatment Mechanism: 2 theories : 1) Antibody-dependent enhancement (ADE) 2) Inherent virulence ADE: Second infection with a heterologous DENV serotype: significantly higher risk of developing DHF and DSS. Pre-existing heterologous dengue antibody recognizes and binds, but does not neutralize the infecting virus and facilitates its internalization via immunoglobulin Fc receptors on the cell membrane of leukocytes, especially macrophages. Prevention: controlling vector mosquitoes - insecticides; water containers etc. vaccine in development Treatment: No specific treatment; supportive therapy - most important is maintaining the circulating fluid volume Chikungunya virus Alphavirus, Togaviridae Virion: 70 nm; enveloped Genome: sS RNA +; 12 kb Transmission: mosquito (Aedes aegypti, albopictus) Prevalence: until recently tropical disease, spreading with the spread of vector Reunion: estimated 312,500 of 757,000 inhabitants infected (2005-07) India: ongoing; 1. 400,000 in 2006 Europe: increasing risk outbreak in Italy, 2007 (A.Albopictus since 1990; scattered foci in almost all regions) Chikungunya virus - continued Infection: self-limiting: fever, headache, weakness, rash, arthralgia (some up to several months even years) 6 - 7 day viraemia Prevention & Treatment No vaccine or preventative drug Measures: Insect repellent containing an DEET or another EPA- registered active ingredient Long sleeves and pants Screens on windows and doors, mosquito net Elimination of mosquito breeding sites (buckets, barrels, tyres etc) BBV pathogenic for certain patient groups Immunocompromised individuals: ~ 50 % transfusion recipients in UK have some degree of immunosuppression Pregnant women: Congenital infection or infection at/shortly after birth - risk of miscarriage, developmental defects CMV Screened for in a proportion of donations. CMV-negative products given to at risk patients B19 → B19 Becoming established. However, ongoing discussion on the introduction of PCR screening. LCMV Several episodes of fatal solid organ transplantations [HHV 6, 8] [Enterovirus ?] Cytomegalovirus (CMV) Cytomegalovirus, Herpesviridae Virion: 120 -150 nm, enveloped Genome: dS DNA, 230 kbp → ~ 200 genes Genotypes: 2 - 6 major subtypes when individual genes typed Variants: Complex population of virus strains (virtually infinite number if sufficient number of genes analysed) Transmission: Person-to-person by direct contact; Minority - shedding in urine, saliva Blood, blood products Organ transplantations Vertical Prevalence: Almost all people exposed to CMV when reaching adulthood Seroprevalence 40 - 90 % CMV - continued Infection & Pathogenesis: Incubation period 3 -12 weeks Lifetime infection; Mostly asymptomatic Minority - infectious mononucleosis-like disease ~ 1 % of newly-born infected: 1 in 10 of these develops significant illness involving nervous system damage or developmental disabilities Immunocompromised: significant morbidity and mortality fever, leucopenia, pneumonia, gastrointestinal problems, impaired graft function Late -stage HIV: encephalitis, retinitis Different strains may interact differently with host’s immune system Co-infection with different strains frequent in immunocompromised, new data suggest also in some 20 % of immunocompetent Treatment: Ganciclovir Cidofovir, Foscarnate - alternatives as the resistant mutants appear Parvovirus B19 Erythrovirus, Parvoviridae Virion: 18 - 26 nm; non-enveloped Genome: sS DNA ~5.4 kb; + and - packaged; 115 nt at the ends → hairpin Codes for VP 1 - 3, NS1, 11kDa protein, 7.5 kDa protein (?) Genotypes: 1-3 Transmission: Respiratory droplets Blood, blood products Prevalence: Seroprevalence: children under 5: ~ 2% 15 year olds: ~ 50% adult population: ~80 % over 60 year olds: >90 % B 19 (continued) Infection: “Fifth disease” Seasonal character: maximum in spring in mild climate countries 108 - 1014 /ml Until recently generally accepted view of self resolving infection within 6 months. New data (NAT) suggest persistence in immunocompromised persons. Possibility of reactivation during other infections and drug treatments Pathogenesis: Immunocompromised individuals: prolonged anaemia, aplastic crisis in individuals with haemolytic anaemia Vertical transmission: Treatment: anaemia, myocarditis → “hydrops fetalis” Generally no treatment required In cases of persistence, severe anaemia - intravenous IgGs LCMV: Lymphocytic choriomeningitis virus Arenavirus, Arenaviridae Virion: 50 - 300 nm; enveloped Genome: sS RNA -; 2 segments: L coding for: polymerase Z (zinc binding) S coding for: NP (nucleoprotein) GPC (glycoprotein precursor → GP1, 2 Strains: Armstrong immunostimulatory, clone 13 - immunosuppressive WE , Pasteur, Traub, UBC [Marseille, MX – Institute of Virology, Bratislava] Transmission: Exposure to rodent aerosolised excreta Organ transplantation: 2 episodes 2003 and 2005 USA 1 episode 2007 Australia 10 out of 11 → deaths [Blood, blood products?] ‘Rare’ person-to-person? LCMV - continued Infection & Pathogenesis: Prevalence: Acute infection usually asymptomatic, self-limiting ‘Grippe-like’: fever, weakness, arthralgia, myalgia, headache, nausea In some cases ‘acute aseptic meningitis’, meningoencephalitis Pregnant women: may lead to abortion, congenital malformations hydrocephalus, macro and microcephaly, chorioenitis Acute infection: NP, GPs well expressed Persistent infection: NP expressed normally, GPs very limited expression Seroprevalence: USA, Canada Italy, Spain Argentina but: Treatment: Slovakia Croatia no specific treatment 4-5% 2-3% 1 - 3.6 % 37.5 % (anti - MX NP) 36 % Pathogen detection Antigen Antibodies (Glyco)protein HBsAg [HCV core] Nucleic acid TARGET AMPLIFICATION HCV RNA HIV1 RNA [HBV DNA] [Bacteria] ELISA, EIA [Combi] Anti-HIV 1,2 Anti-HCV Anti-Treponema Anti-HTLV [Anti-HBc] [Anti-malaria] PCR After J. Mullins (April 01) http://ubik.microbiol.washington.edu/Index.html PCR vs. other amplification techniques Method Target Signal amplification amplification PCR Exponential No Yes High Dispersed LCR No Exponential Yes High Dispersed No High Dispersed NASBA Branched DNA RCA Exponential Thermocycling No Sensitivity Product No Exponential No Medium Localised No No Linear (1 primer) Exponential (2 pr) No No Medium High Localised Dispersed Serological Methods After J. Mullins (April 01) http://ubik.microbiol.washington.edu/Index.html Flat surface microarrays Microparticle based microarrays 1 - 6 μm Carboxylate Avidin-modified Hydrazide Maleimide 75 – 900 μm Solid, split pins Particle encoding/decoding Probes: Nucleic acids, proteins, carbohydrates, cells Surfaces: Poly-L-lys, epoxy-silane, gold, amino-silane… 2-dye scale 100 combinations 100s to 100 000s of spots In situ decoding PCR and microarray PCR Standard Sensitivity Real time High (~10 geq) Multiplexing Limited (30) Confirmation Gel, microarray Protein assays * not required Microarray Low - Medium Very limited (5) No NR* High PCR (for expression profiling) Yes Pathogen blood screening: Examples of assays used in SNBTS Detected target Detection method Instrumental platform HBsAg Sandwich immunoassay Abbott Prism Chemiluminiscence 1-10 pg/ml a) 200/hr Anti-HCV Sandwich immunoassay Abbott Prism Chemiluminiscence NA b) 200/hr Anti-HIV1,2 Sandwich immunoassay Abbott Prism Chemiluminiscence NA b) 200/hr Anti-treponema Agglutination Olympus CCD NA b) 240/hr HCV RNA Real time PCR In-house Fluorescence 29 geq/ml c) 30 –50/6 hrs d) HIV1 RNA Real time PCR In-house Fluorescence 34 geq/ml c) 30 – 50/6 hrs a) b) c) d) Signal readout Detection limit Throughput (No of samples) Estimate Not applicable geq: genome equivalent minipools of 95 sample Modified from: Petrik J, Robb JS: Microarrays and Blood Diagnostics. In:”BioArrays: From Basics to Diagnostics” (K.Appasani ed), Humana Press Inc., Totowa, NJ, 2007; 215-230. New Test Implementation and Declining Risk of Viral Infections from Transfusion Updated from AuBuchon, Birkmeyer, Busch. Ann Intern Med 1997;127:904-9. Risk (per unit) of transmission of major viruses pre-NAT MP-NAT ID-NAT HIV 1 : 1,300,000 1 : 1,900,000 1 : 3,000,000 HCV 1 : 230,000 1 : 1,600,000 1 : 2,300,000 HBV 1 : 180,000 1 : 210,000 1 : 410,000 Summary of risks of transfusion 10 -8 10 -7 -6 10 10 -5 10 -4 10 -3 10 -2 10 General anesthesia HIV HCV HBV Bacteria Mistransfusion Lung injury GVHD Cardiac After S. Dzik, MD Blood Transfusion Service, MGH, Boston Metabolic risk in neonates Under transfusion -1 10 0 Global burden of BBV Cumulative number of infections by HBV, HIV, HCV: 1/10th of world population Mortality related to HIV HBV HCV Chronic infections Cost of HCV therapy Cost of HAART Cost of HBV therapy 2.1 million 1 - 1.2 million ~> 1 million ~£8,500 / year $ 12,000 - 24,000 / year ( less in developing countries) $3,500 (lamivudine) $7,400 - 9,000 (adefovir, entecavir, telbivudine) $24,000 peginterferon Liver transplant $ 100,000 - 400,000 in developed countries $ 18,500 in India Testing > $ 1.3 Billion Individual burden of host - microbes interactions Human cell E coli Vaccinia virus ø 10 μm 0.8 x 2 μm 0.3 x 0.25 μm 1000 pg 0.665 pg 665 fg 0.095 pg 95 fg 1014 1015 Adenovirus ø 0.075 - 0.09 μm 0.00029 pg 0.29 fg >1012/day What is the cumulative “metabolic burden” of simultaneously replicating microorganisms in the host? Levels of complexity of host - microbe interactions 1 Pathogen intraspecies variability Quasispecies: heterogeneous, but closely related swarm of viruses within the same host HCV example: error rate (1 in 10-4-5) and high turnover rate (1012 /day) → → every possible mutation in every position theoretically generated in infected host every day 2 Pathogen - pathogen interactions: Co-infections: HIV-infected individuals: in Europe ~ 9 % co-infected with HBV, 30 % with HCV generally accelerated diseases progress, worsened prognosis HIV + TTV: Increased titres of TTV with progression towards AIDS in serum, bone marrow, spleen etc. HIV + HGV: There seem to be some slowing down effect of HGV coinfection Complex populations of respiratory tract infections 3 Host genomic variability Pharmacogenomics and individualised therapy Prediction of response to particular drugs Individualised dosing regimes 4 Drug interactions Personalised medicine and infectious diseases Concept of “clean” infection with one well-defined pathogen – very simplistic Our data set prior to therapeutic intervention is currently extremely limited Personalised infectious disease therapy will require: Quick analysis of the pathogen populations present, perhaps via coupled PCR - microarray approach: Species identification Genotyping Characterised mutants Quasispecies number estimate if relevant Quantification Host immune system components analysis Host pharmacogenomics predicting drug responses At the same time, continuous surveillance and monitoring of the emerging and reemerging pathogens must take place, resulting in preventative measures and development of vaccines and new therapeutic approaches So there is a lot of work waiting for you – good luck! Thank you for your attention