The viral microbiome

Assoc. Prof. Dr. Christoph Steininger Klinik für Innere Medizin I Medizinische Universität Wien

. Size 1/100 of . Replication only in living cells . High genetic variability (no conserved regions like 16S rRNA) . : RNA & DNA, single- & double-stranded

Course of . Acute (Rhinovirus, ) . Persistent (Lymphocytic choriomeningitis ) . Latent, reactivating (Herpesviruses) . Slow virus infection (HIV, SSPE) Discovery of transmissible tumors

Francis Peyton Rous

1911: Cancer has to be caused by a virus 1961: Identification of Rous Sarcoma Virus as ssRNA virus

Rous Sarcoma Virus 1966: Nobel prize in Physiology/Medicine Cancer associated with human papillomaviruses

High-grade cervical lesions in Australian women aged <18 yrs

Margaret Stanley, Nature 2012

Brotherton et al. Lancet 2011 Merkel cell carcinoma

• Rare, aggressive • Merkel cell polyomavirus (MCPyV) DNA detectable in 80% of MCC tumors • Seroprevalence of polyomavirus in general population: . BK virus 92% . JC virus 45% . WU polyomavirus 98% . KI polyomavirus 90% . MCPyV 59% Carter et al. J Natl Cancer Inst 2009; Pastrana et al. PlosPathogens 2009 Cancer caused by infectious pathogens (Status 2014)

EBV; 5% KSHV; Other; 1% 2%

Helicobacter; HBV & HCV; 32,5% 30%

HPV; 30%

De Martel et al. Lancet Oncology 2012 . 12,7 Mio new cases of cancer in 2008 . 16% of cancer cases attributable to infections . Relevance of infections in carcinogenesis higher in developing countries Estimated burden of chronic viral infections in humans

. ~8–12 chronic viral infections per individuum

Virgin et al. Cell 2009 Diseases of unknown etiology with suspected viral cause

Wylie et al. Curr Opin Microbiol 2013

Virus-host equilibrium in chronic viral infections

Chronic infection - Continuous/intermittent Ag presentation - Tissue damage & aberrant repair - Altered homeostasis - Genotoxicity - Proliferative responses

Cancer

Viral strategies Immune response - Latency - Dampen responses - Immune evasion - Chronic activation - Mutation - Immunopathology - Immunoprivilege - Repertoire contraction Detection of novel oncogenic viruses

Method Issues Cell culture • Selection of cells for permissivity • Identification of Microscopy • Insensitive • Size of viral particles • Differentiation of viruses PCR • highly variable Serology • Cross-reactivity

Adenovirus

Virus attachment Virus culture Electron microscope

Breitbart et al. Res. Microbiol. (2008); Allander et al. Proc. Natl. Acad. Sci. USA (2005); Breitbart et al. J. Bacteriol. (2003); Breitbart et al. Biotechniques (2005); Bibby et al. Trends in Biotechnology 2013 The human microbiome

. Bacteria . Fungi . Parasites & Ova Meiofauna () . Protozoa . Viruses (109 virus particles/gram feces) 1. Eukaryotic Viruses 2. Prokaryotic Viruses (Bakteriophages) 3. Plantviruses 4. Human endogeneous (HERV) ScienceNews

Popgeorgiev et al. Journal of Infectious Diseases 2013; Ogilvie et al. Nat Comm 2013 Plasma DNA Source: 1. Eukaryotic intestinal viruses

. Documented outbreaks of in the US . Caliciviruses . Sapovirus . Enteric Adenoviruses . . Aichivirus

Source: CDC 2014 Discovery of novel eukaryotic viruses

Virus Replication Disease Reservoir Ref. Cyclovirus-Vietnam Feces & CSF Encephalitis Pigs, poultry Van Tan et al. Mbio 2013 Burkina Faso Feces Diarrhea ??? Phan et al. J Clin Astrovirus Virol 2014 Virginia Astrovirus Feces Diarrhea Mammals Finkbeiner et al. J Virol 2009 Torque Teno Serum (HIV- ??? ??? Farsani et al. Journal Mimi Virus 13 positiv) 2013 Humanes Respiratory Bronchilitis Bats, civet cat Van der Hoek et NL63 secretions al. Nat Med 2004 Lujovirus Serum & Tissue Hemorrhagic ??? Briese et al. PlosPathogens fever 2009 Feces Diarrhea ??? Leeuwen et al. J Clin Microbiol 2011 Human Plasma Fever African Green Scuda et al. J polyomavirus 9 Monkey Virol 2011 (HPyV9). Discovery of novel human viruses

Cumulative number of viruses discovered

Arboviruses and non-arboviruses discovered annually

Woolhouse et al. Proc Biol Sci. 2008; Rosenberg et al. PNAS 2013 Transspecies transfer of viruses

Graham et al. Nat Rev Microbiol 2013

. >80 Mio pet cats worldwide . Feline Co-infections common (40% of cats shed >5 viruses in feces) . Novel viruses in asymptomatic cats (Sakobuvirus, Bocavirus, Astroviren, Picobirnavirus)

Ng et al. Veterinary Microbiol 2013; Benedictis et al. Infection, Genetics, Evolution 2011 The „silent“

Group Family Species dsDNA Enteric adenovirus 40, 41 Lymphocystis disease virus phiBCD7, Bacillus phage G, phiP-SSM4 Enterobacteria phage P22, phage T3 Listeria phage A118, phiE125 Lactococcus phage bIL285, phiCP39-O, Clostridium Phages Halophage eHP-10 Human papillomavirus 6, 18, 66 BK virus, JC virus, SV40 virus, Human polyomavirus 9, 12, Merkel cell polyomavirus Epstein-Barr virus, Human ssDNA TTV Chicken anemia virus, TN9, PK5034, PK5222, NG12 Chlamydia phage 1,3,4, Bdellovibrio phage phiMH2K, Chlamydia phage CPG1, Spiroplasma phage 4, Chlamydia phage CPAR39 dsRNA Picobirnaviridae Human picobirnavirus Human rotavirus (+) ssRNA Norwalk virus Astroviridae Human astrovirus Pepper mild mottle virus, Tobacco mosaic virus Picornaviridae Cosavirus, Klassevirus/salivirus, Aichi virus, Enterovirus, Parechovirus, Saffold cardiovirus, Echovirus, , Popgeorgiev et al. Intervirology 2013; Wylie et al. BMC Biology 2014; Foulongne et al. PlosOne 2012 The human megavirome

. Mimi-, Mega-, – infect Acanthamoeba . Human, intestinale Megaviridae . Iridoviridae Acanthamoeba polyphaga sibericum . Poxvirus-related genomic sequences . Senegalvirus . Marseillevirus . Human, Plasma-Megaviridae Poxvirus . Giant Blood Marseillevirus (infects T-cells) E. coli Sputnik . Discovery of Parvovirus Myovirus Mimi- Legendre et al. PNAS 2013; virus Popgeorgiev et al. Intervirology 2013; Popgeorgiev et al. J Infect Dis 2013 Raoult, The Scientist 2014 2. Intestinal

. ~10x more viral particles than bacteria in feces

. High level of genetic diversity . Source of transfer of between bacteria

. Bactericidal due to lytic replication Norman et al. Gastroenterology 2014; Barr et al. PNAS 2013 Phages protect from pathogenic bacteria

Barr et al. PNAS 2013 crAssphage

. 6x more abundant in feces than all other known phages together . 90% of all reads in virus-like particle (VLP)-derived metagenomes . 22% of total community metagenomes . 1.68% of all human fecal metagenomic sequencing reads in the public databases

. Year of discovery: 2014 . Majority of crAssphage-encoded match no known sequences in the database  discarded as data junk

Dutilh et al. Nat Comm 2014 3. Intestinal plant viruses

Pepper mild mottle virus

Zhang et al. Plos Biology 2006; Colson Plos ONE 2010 4. Human endogenous retroviruses (HERV)

. 8%–9% of the human genome EBV infects T-cells . HERVs are mostly replication defective (no virus generated) . Defective HERVs may still IFN-α↑ . express viral proteins . regulate cellular expression Activation of silent . modulate host immune response HERV-K18 element . participate in mutagenic events

Env overexpressed (superantigen activity)

Inflammatory cascade Feschotte & Gilbert, Nat Rev Genetics 2012 initiated Role of HERV-W (syncytin-1) in human cancer

. Overexpressed in 76% of urothelial cell carcinoma tissues (controls, 6%) . Overexpression increases proliferation and viability of immortalized human uroepithelial cells . Involved in mediating cancer-endothelial cell fusions in vitro

. Syncytin-1 expression = positive Urogenital cancer tissue prognostic factor in breast cancer . Syncytin-1 expression in 38% of all breast cancer samples . Upregulated in endometrial carcinoma

Yu et al. 2014; Blesa et al. Cancer Therapy 2008; Larsson et al. Placenta 2002 Physiologic functions of syncytins

. Inserted in human-prehominoids chromosome 7q21, 40-45 millions years ago . Absolute requirement for placenta development and embryo survival

. Positive selective Syncytin gene advantage – switch from capture events egg to uterus . Induction of maternal immune tolerance to fetus? . Cellular resistance against infection by retroviruses of family spleen necrosis virus

Lavialle et al. Phil. Trans. R. Soc. B 2013; Blesa et al. Cancer Therapy 2008 Endogenous viral elements (EVE)

Family or Taxa Number per haploid . Chromosomal genus genome integration of viral Baculovirus Insects Unknown Herpesviridae Humans 1 DNA (or DNA Nudivirus Parasitic wasps Several Brown algae 1 copies of viral RNA) Circoviridae Mammals 1 to 2 in the host germ Tomentosae 5 to 120 Mammals; shrimp 1 to 3 cells Plants; arthropods; Protozoa 1 to 4 Reovirus Aedes spp. mosquitoes 1 . All major types of Fungi; plants; 1 to 6 Honeybees 1 eukaryotic viruses Medaka fish; mosquitoes 1 to 4 can give rise to Grapes Several Vertebrates 1 to 17 EVEs Bunyaviridae Ticks 14 Mammals 1 to 13 Nyavirus Zebrafish 6 Ticks 1 Insects (ticks and mosquitoes) 1 to 28 Retroviridae Vertebrates Several hundreds to several hundreds of Feschotte & Gilbert, Nat thousands Rev Genetics 2012 Hepadnavirus Passerine 15 Pararetrovirus Plants A dozen to a thousand Immune response to the „silent“ virome

Pepper mild mottled virus- specific IgG

Vanura, Steininger et al. PlosOne 2013

Colson et al. PlosOne 2010 Restrained T-cell proliferation in chronic viral infections γ - 2 Viral pathogen IL - CTL TNF IFN potential Apoptosis Proliferative

Cytomegalovirus +++ ++ ++/- +/- ++ - Epstein-Barr virus ++ + + - + - HIV, HCV, HBV +/- +/- +/- - +/- +/-

Murali-Krishna et Chronic LCMV +/- - - - - ++ al. Immunity 1998; Miller et al. Apoptotic T cell +++ Immunity 2008; Virgin et al. Cell 2009 Transkingdom interactions

Opportunistic infections in AIDS patients

CMV Retinitis

Tuberculosis Toxoplasmosis

PML

Kaposi Pneumocystis sarcoma pneumonia Expansion of the intestinal virome during HIV replication

Bacterial microbiom not affected by SIV- infection

Handley et al. Cell 2012; McKenna et al. PLoS Pathogens 2008; Duerkop & Hooper, Nat Rev Immunol 2013; Salzman et al. Nat. Immunol 2010; Fukuda et al. Nature 2011; Scott et al. Cell 2012 Protection of mice from Yersinia pestis and Listeria monocytogenes infection by latent herpesvirus infection

Y. pestis L. monocytogenes

Barton et al. Nature 2007 The parasites of the parasites of the …

Susceptibility to Cervical Vaginal Trichomonas other STI (HIV & & dysbiosis infection HPV) prostate cancer . Metronidazole Treatment: . Parasite clearance . Prognosis for pregnancy and fertility unchanged . Protozoa carry endosymbiotic dsRNA viruses (Trichomonas, Leishmania, Giardia, Plasmodium, Entamoeba, Naegleria, Eimeria, Cryptosporidum, Babesia) . 50% of Trichomonas isolate carry Trichomonasvirus . Nonpathogenic to protozoan host . Contribute to adaptation to and pathogenicity in human host . Metronidazole releases virus from protozoan host  proinflammatory cascade Fichorova et al. PlosOne 2012 Conclusions

 Viral pathogens have been linked to multiple human malignancies

 Underestimated role of viral infection in evolution of human malignancy suspected:

 Kingdom of viruses extensive

 „Silent“ viral infections may be associated with significant inflammatory response

 Extensive interactions between host, virus and other microbes

 Metagenomic studies will very likely open whole new avenues better understanding and targeted therapies in cancer research  Antiviral drugs and vaccines are developed at rapidly increasing pace  Targeted therapies may become available soon Thanks for your attention