Old Herborn University Seminar Monograph 31: Evolutionary biology of the , and impacts in human health and disease. Editors: Peter J. Heidt, Pearay L. Ogra, Mark S. Riddle and Volker Rusch. Old Herborn University Foundation, Herborn, Germany: 5-13 (2017).

EVOLUTIONARY BIOLOGY OF THE VIROME AND IMPACTS ON HUMAN HEALTH AND DISEASE: AN HISTORICAL PERSPECTIVE

PEARAY L. OGRA1 and MARK S. RIDDLE2

1Jacobs School of Medicine and Biomedical Sciences, University at Buffalo, State University of New York, Buffalo, NY, USA; 2Uniformed Services University of Health Sciences, Bethesda, MD, USA

“THE SINGLE BIGGEST THREAT TO MAN’S CONTINUED DOMINANCE ON THE PLANET IS A ” (Joshua Lederberg)

This quote opens the Hollywood movie “Outbreak” to introduce the epidemics of Haemorrhagic Virus fever in Zaire in 1967 and again in the mid 1990’s. The movie is a fascinating commentary on the contemporary perceptions of serious or fatal virus infections, and subsequent impact on human rights and the societal good versus evil. The perception of as evil life forms has been part of human society for thousands of years, and the word “virus” has been used in Latin, Greek and Sanskrit languages for centuries to describe the venom of snake, a dangerous slimy liquid, a fatal poison, or a substance produced in the body as a result of disease, especially one that is capable of infecting others.

Although numerous studies have at- viruses exist per ml of water in the tempted to explain the evolution of vi- oceans. It has been suggested that vi- ruses and their biologic ancestry, the ruses outnumber their hosts globally by precise origins of viruses continue to tenfold or more (Proctor, 1997). Virus remain a mystery. Recent studies have as an infectious entity was initially in- proposed that modern viruses are possi- troduced to describe a process of non- bly derived from multiple ancient (now bacterial pathology by Ivanovsky in extinct) cell types that harboured seg- 1892. In 1898, Beijerinck inde- mented RNA genomes and co-existed pendently applied the term to identify with the ancestors of modern living the causative agent of tobacco mosaic cells (Nasir and Caetano-Anolles, disease (Johnson, 1942). Three decades 2015). Despite the mystery surrounding later, Stanley succeeded in crystalizing their origins, viruses have played a key the tobacco mosaic virus (Stanley, role in the exploration of modern cellu- 1935). The crystalized preparations re- lar and subcellular biologic systems, tained all the biologic properties, in- especially in defining the structure and cluding the ability to infect live cells. function of the genes, and in the explo- As a result, Stanley concluded that the ration of many other aspects of molecu- viruses could not be truly alive. lar biology. Of particular importance Although the existence of viruses as has been the role of viruses in human a distinct morphologic and biologic diseases. entity has now been established for Viruses represent the single most over a century, most modern classifica- successful (numerically) biologic entity tions of existing life forms have to date. It is estimated that >1031 virus continued to exclude viruses from the particles inhabit the earth and over 107 tree of life, in part, because they did not

5 exhibit the ribosomal genes which are genome sequencing, it now appears routinely employed in microbial phylo- that most living organisms may in fact genetic analysis. Recent identification be chimeras containing genes from of a species with dimen- many different sources, including ear- sions and size of the genomes as big as lier eukaryotic life forms, prokaryotic many other microbes, and the discov- unicellular non-nucleated organisms; ery of mimivirus, a parasite of amoeba and subcellular life forms including with a 1.2 megabase-pair DNA ge- viruses. Recently, based on a series of nome, and of other giant viruses whose elegant studies, Forterre and his col- genomes contain over 1.9 to 2.5 million leagues have suggested that all life bases have challenged these percep- forms on earth could be more appropri- tions of the viruses (Raoult et al., 2006; ately divided into two main groups: LaScola et al., 2008; Desnues et al., 1) ribosome encoding organisms (mo- 2012). dern cells) and, Based on ribosomal gene analysis, 2) encoding organisms (vi- the living organisms have now been ruses). classified in the following domains: Forterre has proposed that all modern 1) Subcellular organisms such as cells descend from “the last universal viruses. cellular ancestor or the last universal 2) and prokaryotes, as uni- CenAncester (LUCA). The ribosomes cellular organisms without a nucleus of LUCA contain 33 ribosomal pro- or any membrane bound organelle in teins and 3 rRNA molecules. He and the cell. The classical examples in- his colleagues have proposed that the clude bacteria such as E. coli, modern universal optimized genetic Streptococcus pyogenes, and cyano- code was possibly operational in the bacteria. However, Proteobacteria LUCA. Therefore, it may be possible to are phylogenetically and physiologi- draw a new tree of life connecting to- cally related to eukaryotes. gether all ribosome-encoding organ- 3) Archaea. These resemble bacteria, isms. However, there is not a single are prokaryotes and contain no nu- informational molecule identified to cleus. They make up their own do- date that is common to all viruses (For- main, live in a wide range of ex- terre and Prangishvilli, 2009). Alt- treme environmental conditions. hough, these authors concluded that 4) Eukaryotes. Include virtually all nu- “understanding how modern viruses cleated unicellular or multicellular originated, thus appears to be a more organisms including, fungi, plants, complex problem from the start than animal (including man), and understanding the evolutionary history 5) Protista. These include some mul- of modern cells”, they have also argued ticellular and single cellular organ- strongly in favour of the possibility that isms which don’t fit in other life viruses are essentially parasitic organ- categories. They are characterized in isms which infect living cells and pro- two major taxons; Animal like pro- duce virions in order to spread their tists include amoeba, trypanosomes, genes. Viral genes possibly originated plasmodium (malarial parasites), in the “virosphere” during replication crithidia. Plant like protists include of the virus genome, or were recruited red or green algae and possibly from other cellular lineages which are other still to be classified life forms now extinct (Forterre, 1992). Certain (Woese and Fox, 1977). specific viral proteins are present in the Based on the extensive use of whole existing cellular domains of life forms

6 Table 1: Relative distribution of different viruses in different life forms*. ——————————————————————————————————————— No. of virus genera in the life forms ———————————————————— Virus types Prokaryotic Eukaryotic ——————————————————————————————————————— RNA Viruses Few Many (+) RNA <10 150-160 (-) RNA <2 50-60 dsRNA <2 30-35

Retroviral 5-6 15-20

ssDNA 8-10 >100

dsDNA (All) 40-50 70-80 ——————————————————————————————————————— *Adapted from King, et al., 2011.

infected by different viruses, raising the was “invented” by viruses, helping to distinct possibility that viruses are very convert a world of RNA based organ- ancient, and at least two types of viri- isms to one of DNA-based hereditary ons may have originated independently ancestry, and giant viruses represent before the evolution of LUCA (For- the origin of the nucleus of eukaryotic terre, 1992). More recently, these life forms (Forterre, 2010). investigators have proposed that DNA

ENVIRONMENT AND HUMAN VIROME

Environmental Virome teria and archaea; but no viruses are Viruses inhabit virtually the entire known to be shared by eukaryotes with spectrum of unicellular or multicellular other life forms. Based on the classifi- life forms described above and which cation of viruses introduced by Balti- exist on earth today. However, limita- more nearly 40 years ago, seven classes tions in our ability to detect, isolate and of viruses have now been identified in classify many known and still to be dis- the environmental virome (Baltimore, covered viruses have limited our ability 1971; Koonin et al., 2015). These in- to explore in detail the comprehensive clude: evolution of the global environmental 1) positive stranded RNA viruses, virome. It has been proposed that of the characterized by virions containing over 1031 viral particles present in the RNA of the same polarity as mes- current global microbial population, senger RNA (mRNA), only about 2200 genomes from double 2) negative stranded RNA viruses, stranded DNA viruses (dsDNA) and 3) double stranded (ds)RNA viruses, are well established, as op- 4) reverse transcribing viruses with posed to over 45,000 bacterial genomes positive stand RNA genome, identified to date (Koonin, 2015). 5) reverse transcribing virus with The of the major cellular dsDNA genome, life domains are strikingly different in 6) single stranded DNA (ssDNA) different life forms. Several forms of viruses, and dsDNA viruses are present in both bac- 7) dsDNA viruses.

7 Table 2: Relative distribution of DNA or RNA viral families infecting different life forms. ——————————————————————————————————————— Percent Viral Families —————————————————— Life forms RNA viruses DNA viruses ——————————————————————————————————————— Eukaryotic: Fungi 80 20 Plants 86 14

Animals (invertebrates and vertebrates) 60 40

Protista-like and algae 57 43

Bacteria: Proteobacteria 28 79 Other bacteria 0 100 Archaea 0 100 ———————————————————————————————————————

In prokaryotic organisms most viruses gae) are shown in Table 2 (Letunic and possess dsDNA genomes, with a sig- Bork, 2007). nificant minority of ssDNA containing It is clear that the environmental viruses, and only a very small number virome is immense and virtually all life of RNA viruses. On the other hand, forms are inhabited by viruses. It ap- RNA viruses represent the majority of pears that the genome size of dsDNA virome diversity in Eukaryotic life viruses have the highest diversity and forms, with a significant, although a surpass the far more restricted genome smaller number of ssDNA, and dsDNA sizes of RNA and ssDNA viruses. viruses (King et al., 2011), as shown in However the RNA viruses exhibit a Table 1. It should however be noted broader range of hosts in eukaryotic that genome sizes of RNA viruses are life forms, and infect a lot fewer bacte- much more restricted compared to the rial species when compared to the DNA viruses which exhibit signifi- DNA viruses. cantly more genome size diversity, of- ten over 4 orders of magnitude higher Human virome genome size ranges (Campillo- The human virome represents a diverse Balderas et al., 2015). Of all the known spectrum of viruses found in other pro- viruses, about 9% have 2-3 segments karyotic and eukaryotic life forms and about 27% exhibit more than 4 which inhabit various systemic sites, segments. RNA viruses appear to be skin or the mucosal surfaces in hu- segmented more frequently (50%), than mans. The viral component of the hu- DNA viruses. Of all the segmented man is also referred to as DNA and RNA viruses 60% infect the human virome. plants, 21% infect other phylogenet- The repertoire of viruses in human ically distant plants and other animals virome includes traditional human including 18% vertebrates and, about viruses, bacterial viruses (bacterio- 1% infects bacteria (Campillo-Balderas phage) associated with bacterial species et al., 2015). The relative distribution which constitute the human micro- of RNA or DNA viruses in other eu- biome, and viruses infecting fungi and karyotic life forms, (including, fungi, plants associated with the human plants, animals), bacteria, archaea and microbiome. Protista-like organisms (including al-

8 Table 3: Listing of the viral families in the human virome identified to date. ——————————————————————————————————————— RNA viruses: Viral family: dsRNA ssRNA(+) Coronaviridae; Astroviridae; Calociviridae; Flaviviridae; Picornaviridae; Togaviridae; ssRNA(-) ; Feloviridae; Paramyxoviridae; Arenaviridae; Bunyaviridae; Orthumyxoviridae; Deltavirus.

DNA viruses: Viral family: dsDNA Herpesviridae; ; ; ; ssDNA ;

Retro viruses: Viral family: ssRNA (RT) Retroviridae dsDNA (RT) ———————————————————————————————————————

Human viral infections can manifest as: study viruses now include meta- a) asymptomatic and acute self-limit- genomics analyses, employing com- ing, or as fulminant and progressive parison of genetic information from infections, next generation sequencing of clinical b) chronic symptomatic or asympto- samples to genomes of all known matic infections, viruses, and techniques designed to c) endogenous retroviral infections translate viral genes into proteins and (retroviruses comprise over 8% of computationally search for similar pro- the human genome), and tein sequences in newly discovered d) still to be discovered viruses related agents (Wylie et al., 2012). These ad- to many diseases currently consid- vances have made it possible to charac- ered to being of unknown aetiology. terize rapidly the existing viral ge- The virome of existing bacterial cell nomes and identify new viral agents, as populations is immense, and bacterio- listed in Table 3. phages affect human health, because Viruses have been recovered from they have major influence on bacterial all human mucosal surfaces, skin and cell population, structure, toxin produc- many systemic sites. In recent studies tion and virulence. For example, bacte- of the DNA virome in several thousand riophage communities in the respira- human samples of blood, faecal sam- tory tract of healthy individuals have ples, respiratory and gastrointestinal been found to be unique in each indi- secretions, saliva, milk, urine, and vidual, representing a random and often other body fluids have yielded a wealth transient sampling of the external envi- of information about the distribution, ronment. On the other hand, the bacte- genetic diversity and pathogenicity of riophage communities in patients with human viruses identified to date. They pathologic states, such as cystic fibro- are listed in Table 4 (Pride et al., 2012; sis, were found to be similar to other Delwart, 2016; Moustafa et al., 2017). patients with cystic fibrosis. Such colo- In one study, sequences for 94 different nization is presumably facilitated by viruses were identified in different the similar underlying airway pathol- human blood samples, including for 19 ogy (Willner et al., 2009). human DNA viruses, and Recent technologic advances to RNA viruses (Moustafa et al., 2017).

9 Table 4: Tissue distribution of different viral families in different human body surfaces or secretions. ——————————————————————————————————————— Virus Genetic diversity Tissue distribution Pathogenic or commensal ——————————————————————————————————————— Adenoviridae High GI, Resp., Urine, Blood Both

Anelloviridae High Blood Commensal/Pathogen?

Astrovirus Medium Blood, GI* Both

Flaviviridae Low Blood Both

Herpesviridae Low Blood, Skin Both

Papillomaviridae High Skin Both

Parvoviridae High Blood, GI* Both

Picobirnaviridae High GI* Commensal/Pathogen?

Picornaviridae High Blood, GI*, Resp.**, Skin Both

Polyomaviridae Medium Blood, Resp.** Skin Both ——————————————————————————————————————— GI*: Gastrointestinal tract. Resp.**: respiratory tract. Adapted from: Delwart, 2016; Moustafa et al., 2017; and Pride et al., 2012.

Introduction of has the same individual. The anelloviruses indeed revolutionized the study of exhibit the highest level of genetic human viruses. For example until diversity known in any viral family to recently, only two polyomaviruses date. Most anelloviruses function as were known as human pathogens. commensals and the infections are However, about 13 other human generally asymptomatic. Increasing polyomaviruses have now been identi- amount of anelloviruses in immunosup- fied and some of these have been pressed subjects have been associated implicated in several neurologic or with possible immunologically medi- renal disorders and other pathological ated chronic inflammation and expres- states in immunocompromised hosts. sion of clinical symptoms. However, Some papillomaviruses are also found their role in the pathogenesis of the dis- in asymptomatic healthy skin, while a ease still remains to be clearly defined few can induce anal or cervical cancers (Spandole et al., 2015). (Foulongne et al., 2012; DeCaprio and The spectrum and the load of differ- Garcea, 2013). Another important ent viruses in the human body are truly recent observation relates to the identi- immense. Over 1010-1011 viral parti- fication of a rarely studied ssDNA cles/gram of faeces representing bacte- virus family, the anelloviruses. It riophage predators of bacteria and ar- appears that they may be the most com- chaea, as well as many other viruses mon human viral infections identified have been identified to date in the hu- to date. These viruses have been mans. Furthermore, as pointed out ear- detected in almost 100% of blood sam- lier, endogenous retroviruses constitute ples from human adults. These viruses at least 8% of human DNA (Minot et are acquired shortly after birth and al., 2011, 2012). In spite of their over- multiple strains have been identified in whelming number (possibly billions),

10 as residents of the human body, only munologic functions that were previ- few hundred human viruses have been ously altered by an induced germ-free clearly associated with serious disease status or the use of antibiotics (Kern- in man (Delwart, 2016). bauer et al., 2014). In another recent Available epidemiologic evidence study, it has been demonstrated that suggests that the acquisition of the early enteric infections can influence environmental virome by mankind is the development of the gut and murine an evolutionary (and possibly a requi- in a very similar man- site) adaptation, and the organisms are ner as observed with the bacterial neither consistently commensal nor microbiome on development of the hu- pathogenic. The outcome of the micro- man gut and maturation of the immune biome-host interaction at a cellular system (Caldwell, 2015). level is determined by a complex bal- Finally, the retroviral components of ance between the pathogenic potential the human genome have also been of the virus and the immunologic status adapted to enhance functional develop- of the host at the time of their initial ment as well as the survival of the and subsequent interactions. mammalian host. It has been proposed Maternally derived antibodies ac- that proteins expressed by such quired transplacentally or via breast endogenous retroviruses can effectively milk induce significant protection bind or block receptors for other exoge- against disease against a variety of bac- nous retroviruses (Griffiths, 2001; Mal- terial as well as viral infections in the favon-Borja and Feschotte, 2015). In neonatal period and early infancy. an elegant study, endogenous retroviral Acquisition of such viral infections envelope proteins were found to be re- during early years of human growth sponsible for the fusion of foetal and development appear to play a very trophoblast cells with the mammalian important role in the maturation and placental cells during the onset of preg- functional development of the host im- nancy. Such fusion is essential for mune system at mucosal and systemic nutritional exchange between maternal sites. Such early infections may also and foetal system during gestation (Mi protect against more pathogenic infec- et al., 2000). tions with other viruses later in life. For Based on the information summa- example, there is evidence to suggest rized above, it is clear that we are just that pegivirus or GBV-C virus belong- beginning to explore and define the ing to the larger family of viruses for- complexity of human virome relative to merly known as of hepatitis G or the immense load of environmental HPgV, dengue and zika viruses can virome. Our understanding of viruses significantly improve the outcome of has evolved as a result of specific HIV infection. Pegivirus-c infection infections associated with many appears to be quite common and eukaryotic life forms, beginning with asymptomatic. It is estimated that to the discovery of tobacco mosaic date over 700 million humans are in- disease virus. However, the human fected with the virus. HIV infected sub- virome, as we understand it today, is jects with pegivirus-c infection tend to immensely larger than the few hundred live longer and experience healthier disease producing viruses. While some lives (Linen et al., 1996) Similarly, ex- viruses are prone to produce acute, perimental animal studies have demon- chronic, fulminant or fatal infections in strated that mouse norovirus, a com- man (Hulo et al., 2011), the human mon human commensal, restored im- virome predominantly represents a

11 large reservoir of symbiotic interac- on human health, immunologic homeo- tions between the viruses and the stasis, and maternal-neonatal interac- human and other life forms on earth. In tions, than the development of serious fact, the human virome may provide or fatal disease. significantly far more beneficial effects

LITERATURE

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