UNIVERSITY OF PÉCS

Doctoral School of Biology and Sportbiology

Evolutionary relationships of bat-related viruses of European bats

PhD Thesis

GÁBOR KEMENESI

PÉCS, 2018 UNIVERSITY OF PÉCS

Doctoral School of Biology and Sportbiology

Evolutionary relationships of bat-related viruses of European bats

PhD Thesis

GÁBOR KEMENESI

Supervisor: Ferenc Jakab PhD, habil

______Supervisor Head of the doctoral school

PÉCS, 2018

- 2 -

Table of Contents

ABBREVIATIONS - 5 -

GENERAL INTRODUCTION - 6 -

INTRODUCTION OF VIRUS GROUPS DESCRIBED IN THE THESIS IN RELATION TO BATS - 10 -

ASTROVIRIDAE - 10 - CALICIVIRIDAE - 11 - CARMOTETRAVIRIDAE - 12 - CORONAVIRIDAE - 12 - MISCHIVIRUS GENUS, FAMILY PICORNAVIRIDAE - 13 - ROTAVIRUS GENUS, FAMILY REOVIRIDAE - 14 - PARVOVIRIDAE - 15 - CIRCOVIRIDAE - 16 - GENOMOVIRIDAE - 17 - FILOVIRIDAE - 17 -

OBJECTIVES - 19 -

MATERIALS AND METHODS - 20 -

SAMPLE COLLECTION - 20 - NUCLEIC ACID PREPARATION - 23 - POLYMERASE CHAIN REACTION (PCR) METHODS - 23 - DETERMINATION OF THE TERMINUS OF GENOMIC RNA - 25 - CLONING AND SANGER SEQUENCING - 26 - NEXT-GENERATION SEQUENCING WITH ION TORRENT PGM - 26 - RECOMBINATION ANALYSES - 27 - PHYLOGENETIC ANALYSES - 27 - PROTEIN MODELING - 27 - NUCLEOTIDE COMPOSITION ANALYSIS - 28 - PAIRWISE IDENTITY CALCULATION - 28 -

RESULTS AND DISCUSSION - 29 -

ASTROVIRIDAE - 29 - CALICIVIRIDAE - 31 - CARMOTETRAVIRIDAE - 39 - CORONAVIRIDAE - 40 - PICORNAVIRIDAE - 42 -

- 3 -

REOVIRIDAE - 48 - PARVOVIRIDAE - 53 - CRESS DNA VIRUSES (GENOMOVIRIDAE AND CIRCOVIRIDAE) - 57 - FILOVIRIDAE - 65 -

CONCLUDING REMARKS - 68 -

REFERENCES - 69 -

ACKNOWLEDGEMENTS - 82 -

SUPPLEMENTARY MATERIAL - 83 -

SUMMARY OF POSITIVE BAT SPECIES PER REGION. - 83 - TWO DIRECTIONAL PAIRWISE IDENTITY MATRIX OF CIRCOVIRUS SEQUENCES - 84 - STRAIN-SPECIFIC OLIGONUCLEOTIDES - 85 - NUCLEOTIDE COMPOSITION ANALYSIS (NCA) RAW DATA - 87 -

LIST OF PUBLICATIONS - 88 -

PUBLICATIONS RELATED TO THESIS TOPIC - 88 - CONFERENCE PROCEEDINGS RELATED TO THESIS TOPIC - 88 - PUBLICATIONS OUTSIDE TO THESIS TOPIC - 89 - CONFERENCE PROCEEDINGS OUTSIDE THESIS TOPIC - 90 -

STATEMENT - 91 -

- 4 -

Abbreviations

AstV – Astrovirus BSL-4 – Biosafety level 4 BtAstV – Bat astrovirus BtCalV – Bat calicivirus BtCoV – Bat coronavirus BtPV – Bat parvovirus CalV – Calicivirus CiV – Circovirus CoV – Coronavirus CoSV – cosavirus CP – capsid protein CRESS DNA – circular replicase encoding single-stranded DNA viruses EBOV – Ebola virus EMCV - Encephalomyocarditis Virus FCV – Feline coronavirus FiV – Filovirus FMDV - Foot-and-mouth disease virus GPS – global positioning system iPCR – inverse polymerase chain reaction IRES – Internal ribosome entry site LLOV – Lloviu virus MARV – Marburgvirus MERS – Middle Eastern respiratory syndrome NCA – Nucleotide composition analysis NPC1 - Niemann-Pick C1 receptor NSP – non-structural protein ORF – open reading frame PBS – phosphate buffered saline PCR – Polymerase chain reaction PiV – Picornavirus PMP – putative middle protein PrV – Providence virus RdRp – RNA-dependent RNA-polymerase RT-PCR- Reverse transcription polymerase chain reaction RV – Rotavirus RVA-I – Rotavirus genotype A-I SARS –Severe acute respiratory syndrome UTR – untranslated region VP – viral protein

- 5 -

General introduction With over 1250 species, bats represent more than 20% of extant mammals, furthermore constituting the most widespread mammalian order worldwide. The order Chiroptera is classified into suborders Yinpterochiroptera and Yangochiroptera, which include biologically and ecologically diverse species that are distributed in all continents except Antarctica (Teeling et al., 2005). Phylogenetic diversity of bats has been widely discussed in several previous studies (Jones et al., 2002; Agnarsson et al., 2011), and the evolutionary historical reconstruction originates this mammalian order in the early Eocene, 50 to 52 million years ago. Their appearance was coincidental with a significant global rise in temperature, increase in plant diversity and abundance, and the zenith of Tertiary insect diversity (Teeling et al., 2005). The approximately long evolutionary history along with unique adaptations among terrestrial mammals, such as echolocation and flight (Wang et al., 2011) resulted in outstanding species diversity and geographic distribution. Bats belong to the most widely distributed land mammals, only humans have reached higher prevalence rates (Wimsatt, 1970). Furthermore, their long lifespan, low rate of tumorigenesis, and an ability to asymptomatically carry and disseminate highly pathogenic viruses (Wang et al., 2011) make them an important investigation target for several research areas, such as immunology, evolution biology or microbiology. Based on their evolutionary history, which resulted in numerous coevolution events, bats and their parasites - including viruses are increasingly investigated for their role in the maintenance of potentially emerging pathogens in nature (Tortosa et al., 2013). A high proportion of emerging and re-emerging infectious diseases are zoonoses derived from wildlife (Jones et al., 2008). Based on a recent study wild animals, which include a taxonomically diverse range of thousands of species (e.g. bats or rodents), were significantly more likely to be a source for animal-to-human spillover of viruses than domesticated species (Johnson et al., 2015). In the last decades, bats were increasingly recognized as the source of emerging diseases which were seriously affected human populations. Henipaviruses (Hendra and Nipah viruses), coronaviruses (SARS and MERS coronaviruses), filoviruses (Ebola virus), and several lyssaviruses (Rabies, European bat lyssavirus 1 and 2, etc.) have all been shown to be transmissible from bats to humans - often through an intermediate host - with fatal consequences (Wynne et al., 2013). Based on the study of Luis and others, bats harbour more zoonotic viruses per species than rodents and are now recognized as a significant source of zoonotic agents (Luis et al., 2013).

- 6 -

The first recognized zoonotic viruses of bats were different lyssaviruses, and now bats are considered as the ancestral host for this group of viruses in carnivores and bats (Badrane et al., 2001; Streicker et al., 2010). Multiple events of last decades pointed out the role of bats as a risk for public health. The emergence of Severe Acute Respiratory Syndrome (SARS) coronavirus in 2002 and Middle Eastern Respiratory Syndrome (MERS) coronavirus in 2012 marked two introductions of a highly pathogenic coronavirus into the human population in the twenty-first century (de Wit et al., 2016). Bat origins were proved and supposed respectively for the two coronaviruses (Lau et al., 2005; Mohd et al., 2016). The West African massive Ebola virus outbreak in 2014, which resulted in over 28 000 human cases with 11 000 fatalities has also been supposed to have zoonotic origins in Angolan free-tailed bats (Mops condylurus) (Mari Saéz et al., 2014). These examples well represent the risk for human health in association with bat-harboured viruses. The risk for the occurrence of these spillover events is highly facilitated by multiple human-animal interactions, which are presented in Figure 1.

Figure 1. Interactions between bats and humans and the possible causes of spillover events (Smith et al., 2013).

- 7 -

Beyond public health issues related to bats, their role in the evolution of several viral families is substantial and extensively examined (Hayman, 2016). For instance, Hepadnaviruses have been associated with old mammalian lineages like bats for a prolonged time. Bat-related hepadnavirus diversity seems to be exceeding the diversity of strains in other host orders, which suggests a long co-evolution past as recently reviewed (Rasche et al., 2016). Additionally, Hepatitis A virus origin was also linked to small mammals, such as rodents and bats and various diverse strains are represented in wide geographic regions among these animals (Drexler et al., 2015). Several other viral families are remarkably diversified in bats, such as Astroviruses, Coronaviruses or Picornaviruses, with distantly related viral strains in broad geographic regions worldwide (Hayman, 2016A). Numerous other viral families are known to be represented by bats: Adenoviridae, Bunyaviridae, Hepadnaviridae, Hepeviridae, Herpesviridae, Papillomaviridae, Paramyxoviridae, Polyomaviridae, Reoviridae, Retroviridae, Caliciviridae, Circoviridae, Rhabdoviridae, Astroviridae, Flaviviridae, Coronaviridae, Picornaviridae, Orthomyxoviridae, and Parvovirinae. Insect viruses from Baculoviridae, Iflaviridae, Dicistroviridae, Tetraviridae, and Densovirinae. Fungal viruses from Chrysoviridae, Hypoviridae, Partitiviridae, and Totiviridae; and phages from Caudovirales, Inoviridae, and Microviridae (Wu et al., 2012; Wu et al., 2015). True host origins of these viruses reported from bats are largely uncertain. Deciphering the true host origins and pathogenic potential of these viruses is challenging and requires further studies. For instance, Providence virus a member of the family Tetraviridae, originally known as an insect virus which persistently infecting a midgut cell line derived from the corn earworm (Helicoverpa zea) (Pringle et al., 2013) was recently described with the possible human pathogenic capability (Jiwaji et al., 2016). This raises the challenge for assessing possible host organisms and emphasizes the importance of in vitro and in vivo experiments beyond massive sequencing procedures. Despite the enormous number of viruses reported in bat populations, many open questions remain about transmission, spillover events, within-host, population, metapopulation and community dynamics. Bats seem to have a propensity to host viruses, yet only a few agents were confirmed as causal organisms implicated in mortality events of bats (O‘Shea et al., 2016). Considering these things, bats represent an important model species for studying the evolution of antiviral immunity. Based on comparative genomic studies, several immune-related genomic changes were described in bats (Zhang et al., 2013).

- 8 -

Other factors affecting immunity were also suggested for the longterm persistence of viruses without symptoms, such as torpor use in case of Rabies (Hayman, 2016 B). Innate immunity-related genes were found to be under strong positive selection in Pteropus alecto and Myotis davidii genomes compared to their ortholog mammalian species (Zhang et al., 2013). Bats and viruses have undoubtedly coevolved over millions of years, and considering previous studies the genetic arms-race between bats and viruses may have reached an equilibrium in case of most bat-harboured viruses, resulting in unique diversity of bats and their viruses. In summary, bats are representing a major gene pool for several known and yet undiscovered viral taxa, from which several may possibly provide the basis of zoonotic events, as described in multiple cases (e.g: Ebola, SARS - and MERS coronavirus) with similar transmission scenarios (Fig. 2.).

Figure 2. Pandemic properties of zoonotic viruses that spill over from animals to humans and spread by secondary transmission among humans (Johnson et al., 2015).

- 9 -

Introduction of virus groups described in the thesis in relation to bats

Astroviridae Astroviruses are taxonomically grouped to the Astroviridae family, which is divided into two genera: Mamastrovirus with 19 accepted taxonomic species and Avastrovirus with three species (Adams et al., 2017). The family comprises a diverse group of small, non- enveloped single-stranded RNA viruses of positive polarity with a characteristic star-shaped appearance. Astroviruses were first discovered in faeces samples of infants suffering from diarrhoea in 1975 (Appleton & Higgins, 1975), since then they are recognized as major causative agents of gastroenteritic infections worldwide. Along with humans, numerous animal species are known to be susceptible to Astrovirus infections, including domestic, synanthropic and wild animals, avian, and mammalian species in the terrestrial and aquatic environments also (Paola et al., 2011). The veterinary and human health significance of astroviruses is well highlighted with the most common clinical picture of diarrhoea or in some cases other, more serious clinical manifestations, such as nephritis, hepatitis or even encephalitis (De Benedictis et al., 2011; Quan et al., 2010). On the other hand, astroviruses are also represent a unique evolutionary diversity, which is mostly associated with various bat species across the globe (Fischer et al., 2017). The first studies which pointed out the outstanding genetic diversity of bat-related astroviruses were conducted in China (Chu et al., 2008; Zhu et al., 2009). Several further studies were performed in China using viral metagenomics and consensus PCR screening. High diversity of members within Mamastrovirus genus of the family Astroviridae with several potential novel species and high prevalence were observed (Li et al., 2010; Xiao et al., 2011; Wu et al., 2012). The first European study was conducted in order to examine the possible connection between bat colony status and virus prevalence variations of adeno-, astro- and coronaviruses. It provided the first sequence dataset of astroviruses circulating among European bat species, although only Myotis myotis individuals were tested (Drexler et al., 2012). Large-scale surveillance studies were performed in China before, in order to reveal Astrovirus diversity of bats (Chu et al., 2008; Zhu et al., 2009). Several novel genetic lineages were discovered with probable host specificity.

- 10 -

Following these studies, genetically related strains were described from the European region in Germany and the Czech Republic, reporting high host specificity of astroviruses in various bat species (Dufkova et al., 2015; Fischer et al., 2016). In order to assess the zoonotic potential of bat astroviruses, further surveillance studies are needed on infection dynamics, shedding and transmission of viruses between bats and potentially from bats to other mammalian species. Although based on our recent knowledge the risk of astrovirus transmission from bats to humans is considered to be rather low, the role of bats in astrovirus evolution and diversity is substantial. The role of bats in astrovirus evolution is most likely associated with their reservoir role, since no disease of infected bats were reported to date.

Caliciviridae The family Caliciviridae comprises small non-enveloped RNA viruses, which are classified in five genera: Vesivirus, Lagovirus, Norovirus, Sapovirus and Nebovirus. In addition, unclassified caliciviruses have been identified in monkeys, pigs, avian species and fishes (Farkas et al., 2008; L’Homme et al., 2009; Wolf et al., 2011; Liao et al., 2014; Mikalsen et al., 2014). Bat-related Caliciviruses were only reported from Hong Kong and New Zealand (Tse et al., 2012; Wang et al., 2015). The common characteristic of these bat-derived calicivirus sequences is the phylogenetic divergence from other known members of the family Caliciviridae. Revealing partial or full-length genome sequence data of these recently discovered viruses is crucial to facilitate viral taxonomical classification, develop reliable diagnostic assays and to better understand the molecular mechanisms driving the evolution and host species adaptation of caliciviruses. Human and animal caliciviruses may evolve by accumulation of punctate mutations and by exchange of genetic material via recombination (Giammanco et al., 2013). Also, the interspecies transmission has been documented repeatedly and the origin of some human calicivirus strains may have been triggered by a combination of various evolutionary mechanisms (Smith et al., 1998; Scheuer et al., 2013). In this regard, the possible reservoir role of bats has been investigated minimally. In addition interspecies transmission of bat-harboured caliciviruses was not reported to date, although this topic was investigated minimally.

- 11 -

Carmotetraviridae Tetraviruses are classified into three families: Alphatetraviridae, Permutotetraviridae, and Carmotetraviridae, according to the sequence of their viral replicase, which may be alphavirus-like, picornavirus-like, or carmovirus-like, respectively. Little is known about the function, evolution and role of this group of viruses. An intensively investigated Tetravirus, Providence virus (PrV) belongs to the genus Alphacarmotetravirus and represents the only member within the family Carmotetraviridae (Adams et al., 2017). PrV was originally isolated from a persistently infected Helicoverpa zeae MG8 midgut tissue culture cell line and is the only tetravirus to replicate in tissue culture (Pringle et al., 2003). The virus has a positive- sense, single-stranded RNA (ssRNA) genome of 6.4 kb encoding three open reading frames (ORFs), comprising the putative viral replicase (p104) followed by the capsid protein precursor ORF, which is translated from a subgenomic RNA of 2.4 kb. Among tetraviruses, PrV possesses a third ORF at the 5′ end encoding a nonstructural protein of unknown function, overlapping with the replicase gene (Walter et al., 2010). Although PrV is an arthropod infecting virus, genomic characterization of geographically distant strains can facilitate the taxonomical and evolutionary investigations regarding this unique viral family. Moreover, the Old World cotton bollworm Helicoverpa armigera, is a close relative of its original host, representing a major pest of agriculture in Asia, Europe, Africa and Australia, such investigations can facilitate future biological control efforts. Providence virus was only described in the United States to date (Pringle et al., 2003). Interestingly Providence virus was reported as a possible human pathogenic virus, based on in vitro experiments (Jiwaji et al., 2016), which raises the importance of this group of viruses for human health aspects also.

Coronaviridae Coronaviruses are classified within Nidovirales order, Coronaviridae family and four genera Alphacoronavirus, Betacoronavirus, Deltacoronavirus and Gammacoronavirus. Alpha – and Betacoronaviruses are mainly infecting mammals, and are reported in association with bats. The emergence of two novel coronaviruses (Severe Acute Respiratory Syndrome - SARS and Middle Eastern Respiratory Syndrome Coronaviruses - MERS) in the human population, causing unexpected human disease outbreaks in the 21st century, well reflected the importance of bats, as a major evolutionary gene pool for these viruses (Hu et al., 2015; Adams et al., 2017).

- 12 -

Following the Severe Acute Respiratory Syndrome (SARS) coronavirus pandemic in 2003, bat-related coronaviruses were extensively examined worldwide. The evolutionary origins of the pandemic strain were clearly connected to SARS-like betacoronaviruses harboured by Chinese Horseshoe bats (Rhinolophus sinicus Rhinolophus pearsonii, Rhinolophus ferrumequinum, Rhinolophus macrotis and Rhinolophus pusillus) (Lau et al., 2005; Ren et al., 2006; Hon et al., 2008). Numerous further studies described SARS-related betacoronaviruses in bats from Asia (Chen et al., 2016; He et al., 2014; Yang et al., 2013; Yang et al., 2015), and Europe (Drexler et al., 2010; Lelli et al., 2013), and now bats are considered as natural reservoirs for SARS-related coronaviruses worldwide (Hu et al., 2015). Along with Astroviruses, Coronaviruses may also present in bat colonies with high prevalence, whereas their role as bat pathogens is still unclear. Additional other aspects of coronaviruses are also poorly examined to date, such as the transmission routes between individuals of the colony (Drexler et al., 2012). Coronaviruses of animal origin were responsible for SARS and recently for current epidemics of Middle Eastern Respiratory Syndrome (MERS) in the Arabian Peninsula and Korea (Choi, 2015; Alsahafi & Cheng, 2016; Anthony et al., 2017). The zoonotic potential of several bat-related coronaviruses is well examined and now seems to be substantial (Hu et al., 2015; Menachery et al., 2015). More interestingly, a cross-family recombinant, bat- originated Coronavirus was recently described, which contained a most likely bat orthoreovirus originated gene with a functional role during virus infection (Huang et al., 2016). The discovery of this virus further highlights the importance of bats as a major gene pool for Coronaviruses and emphasizes the complexity of mechanisms facilitating the evolution of these viruses.

Mischivirus genus, family Picornaviridae Picornaviruses are small, enveloped viruses with a single-stranded, positive-sense RNA genome. The family Picornaviridae belongs to the order Picornavirales and currently consists of 54 species grouped into 31 genera (Knowles et al., 2012; Adams et al., 2017). Picornaviruses are found in humans and a wide variety of animals, in which they have the ability to cause respiratory, cardiac, hepatic, neurological, mucocutaneous and systematic diseases with different levels of severity (Tracy et al., 2006).

- 13 -

Moreover, they assumed to have the potential to cross species barriers, since related strains of rodent-associated Picornaviruses have recently been described in humans and seroepidemiological evidence was found for human infection with heterologous porcine picornavirus (Yu et al., 2013; Lim et al., 2014). Among bat-harboured viruses, members of the Picornaviridae family are represented in high diversity and wide geographic distribution. Three genera of Hepatovirus, Mischivirus and Sapelovirus have been reported to date from bats (Lau et al., 2011; Wu et al., 2012; Drexler et al., 2015). Evolutionary ancestral hepatoviruses are widely present in bats worldwide, although human-infecting hepatoviruses are assumed to be originating from rodent hosts (Drexler et al., 2015). Members of Mischivirus genus were only described in bats to date. One species of Mischivirus A and two proposed species of Mischivirus B and Mischivirus C (African bat icavirus A) are included in the genus. Mischivirus A and B were detected in Chinese and Hungarian Miniopterus schreibersii bats respectively, while Mischivirus C was detected in Hipposideros gigas bats from Congo (Wu et al, 2012; Kemenesi et al., 2015A; GenBank Accession: KP100644). Further unpublished sequence data are available in GenBank database, suggesting a wide geographic distribution in Europe and Asia of bat- harboured picornaviruses with high genetic diversity. The evolutionary pathways, origins and diversity of bat-related Picornaviruses are largely unknown, although the most examined group with a significant evolutionary connection to bats is Mischivirus genus. Available data about these viruses is mainly genomic, and little is known about their possible pathogenic role in bats or in other species as well.

Rotavirus genus, family Reoviridae Reoviridae viral family includes a variety of viral taxa, including Rotavirus (RV) genus Rotaviruses (family Reoviridae; subfamily Sedoreovirinae; genus Rotavirus) which represents the core topic of this paragraph. At present, Rotavirus genus contains 9 accepted viral species, RVA to RVI (Adams et al., 2017). The classification of genetically distinct species within the Rotavirus genus is based on VP6 gene amino acid sequences, with a threshold of 53% identity (Matthijnssens et al., 2012). These non-enveloped viruses have an RNA genome, which is approximately 18.5 kb in size and consists of 11 segments encoding six structural proteins (VP1-VP4, VP6 and VP7) and six nonstructural proteins (NSP1-NSP6) (Estes & Kapikian, 2007).

- 14 -

Generally, Rotaviruses are major causative agents of severe diarrhoea and intestinal distress in children and are able to cause acute diarrhoea in mammals and birds (Estes et al., 2007). Among accepted RV species, RVA to RVC, RVE, RVH and RVI are known to infect mammals and RVA is the most widespread species in most mammalian hosts (Mihalov- Kovács et al., 2015). Bat-related RVs described so far belong almost exclusively to RVA; sequence analysis of the identified strains uncovered some intriguing details concerning the ecology and evolution of bat-harboured RVAs. Reassortment event was described in the case of a RVA strain from Kenya, which possessed an unusual VP1 gene and hypothesis arose the first time that during their evolution mammalian RVs belonging to different RV species may share genes (Esona et al., 2010). Furthermore, bats seem to serve as reservoirs of multiple RVA genotypes commonly found in heterologous host species (Asano et al., 2016). Consequently, bat-related RVAs might pose a veterinary and public health risk. More recent data indicate that in addition to RVA, RVH may also infect bats, although this observation is based on relatively short sequence data described from an undetermined bat species in Korea (Kim et al., 2016). Summarizing all related data, bat-associated rotaviruses are still poorly examined, although they are increasingly reported from various bat species worldwide, raising the assumption that bats may serve as an important evolutionary gene pool for RVs.

Parvoviridae The family Parvoviridae comprises non-enveloped viruses with a single-stranded DNA genome of approximately 5 kb. The subfamily Densovirinae includes only arthropod-infecting viruses, while Parvovirinae contains human and other vertebrate-infecting viruses. The Parvovirinae subfamily is currently subdivided into 8 genera (Cotmore et al., 2014). Among DNA viruses, parvoviruses exhibit the highest mutation rate, almost comparable to that possessed by RNA viruses, furthermore, they are extremely prone to recombination (Shackelton et al., 2007; Decaro et al., 2009). Parvoviruses are associated with a wide spectrum of acute and chronic diseases in humans and animals, with at least five known groups (Dependoviruses, Parvovirus B19, Human bocaviruses, Parv4 viruses and recently described Bufaviruses) infecting humans (Brown, 2010; Phan et al., 2012).

- 15 -

The discovery of human bufavirus in 2012 from faecal samples of children with acute diarrhoea revealed a possible novel human-infecting parvovirus to science (Phan et al., 2012). Since then bufaviruses were further detected in human samples from various geographic regions worldwide (Smits et al., 2014; Väisänen et al., 2014; Yahiro et al., 2014; Altay et al., 2015; Chieochasin et al., 2015; Huang et al., 2015). The evolutionary origin of this newly recognized virus is still unknown, although bufaviruses were discovered in nonhuman primate, rodent and porcine faecal samples (Sasaki et al., 2015; Hargitai et al, 2016; Liu et al., 2016; Sasaki et al., 2016; Yang et al., 2016).

Circoviridae The family Circoviridae incorporates small, non-enveloped viruses with single- stranded circular DNA genome ranging 1.7-3 kb in size (Biagini et al., 2011). Based on recent International Committee on Taxonomy of Viruses (ADAMS ET AL., 2017) classification, two genera, Circovirus and Cyclovirus are currently recognized within the Circoviridae family (Rosario et al., 2017). A proposal was also published to include Krikovirus genus in the family (Garigliany et al, 2015). These circular replication-associated protein encoding single- stranded (CRESS) DNA viruses encode a replication-associated protein (Rep) which is essential for initiating rolling circle replication (RCR). Two main conserved motif categories exist within the Rep, which are important domains for CRESS viruses and molecules to replicate through rolling-circle replication. These categories are the RCR motif I-III and the superfamily 3 (SF3) helicase motifs known as Walker-A, Walker-B and motif C (Rosario et al., 2012). In the case of European bat fauna, dietary viruses mainly incorporate arthropod-related viruses but also reflect other viral sequences which are present in the food chain (e.g. plant viruses). Based on the results of virome analyses of bats in China, CRESS DNA viruses, along with members of the family Parvoviridae constitute the main groups of DNA viruses within bat virome (Wu et al., 2016). In recent years, several novel highly divergent circular single- stranded DNA (ssDNA) viruses were described in bats from Brazil (Lima et al., 2015A; Lima et al., 2015B), China (Li et al, 2010; Ge et al., 2011; Wu et al., 2016), USA (Li et al., 2011A), and Tonga, Oceania (Male et al., 2016).

- 16 -

Over recent decades, metagenomic studies of bat samples along with surveillance studies using degenerate, group-specific primers have expanded the number of newly described, often unclassified viruses within the family Circoviridae (Li et al., 2010; Ge et al., 2011; Lima et al., 2015A; Lima et al., 2015B; Wu et al, 2016). These results may highlight the role of bats as good environmental genomic indicators by representing a wide range of viruses in their excreta, and raise the idea if bats serve as an important host and gene pool for CRESS DNA viruses also. Genomic data of these viruses from European bat species was not described to date.

Genomoviridae Another major group of CRESS DNA viruses are members of the recently established Genomoviridae viral family, and they are widely represented in a variety of environmental, plant and animal samples worldwide. Genomoviridae includes viruses with ~2-2.4 kb circular ssDNA genomes, encoding a rolling-circle replication initiation protein and a viral capsid protein (Varsani & Krupovic, 2017). These circular replication associated protein-encoding DNA viruses seem to be widely present in the ecosystem, including bat faecal samples (Male et al., 2016; Krupovic et al., 2016). The possible role of Gemycircularviruses in human infections was also suggested in a case of unexplained child encephalitis, where a novel member of the genus was detected in the cerebrospinal fluid of the patient (Zhou et al., 2015). Further observations were published regarding the potential human pathogenic capability, since novel Gemycircularviruses were described in association with unexplained central nervous system infections and unexplained cases of diarrhoea, from Sri Lanka and Nicaragua respectively (Phan et al., 2015). The possible pathogenic role of bat-related strains was not proved yet in association with human nor in veterinary infections, in addition their exact host is still unknown.

Filoviridae Members of the Filoviridae viral family such as Marburg virus (MARV) or Ebola viruses (EBOV) represent major public health importance worldwide as a causative agent of Ebola virus disease and Marburg virus disease. Ebola virus is responsible for one of the most devastating epidemic of modern human history that involved more than 28 thousand people, with a mortality rate of 40% during the 2014 West African outbreak (Coltart et al., 2017).

- 17 -

Identification of genetically diverse filoviruses in Chinese bat samples revealed an unexpected genetic diversity and distribution of these viruses (He et al., 2015; Yang et al., 2017). Since the discovery of Lloviou virus (LLOV - pronounced j’ɔːvju vɑɪrəs) in Miniopterus schreibersii samples from Spain, Europe is considered as a potential geographic region for filoviruses as well (Negredo et al., 2011). LLOV is a sole member within Cuevavirus genus, and since its emergence in 2002, no further detection was reported in the world. Moreover the failure of in vitro isolation and complete genomic characterization of the original virus, studies were greatly restricted and were mainly associated with recombinant virus experiments (Burk et al., 2016). The potential of LLOV to infect humans is still unclear, whereas several recombinant LLOV protein studies suggest the potential to infect human and bat cells, using the same receptor entry mechanisms (universal endosomal ebolavirus receptor – NPC1) as Ebola virus (Burk et al., 2016). Because of the absence of a replicating LLOV isolate, no animal model of LLOV infection is available. The possible connection of LLOV with the Spanish mass mortality event of Miniopterus bats suggest the existence of a yet undiscovered natural reservoir of the virus.

- 18 -

Objectives

A major goal of our study is to fill the knowledge gap regarding the viral diversity of European bat species and reveal possible evolutionary connections with distant relatives. As a result of mass sampling of various habitats in large geographic area, we aim to identify key bat species for viral evolution and identify potential pathogens for bat conservation biology efforts as well. Moreover, based on the genomic characterization of these agents we plan to sketch the evolutionary linkages with cognate viruses and provide data regarding the evolutionary history of several viral taxa. Considering the growing public health significance of bat-harboured viruses worldwide, we plan to identify viruses with potential zoonotic spillover ability as well.

In summary:  Large-scale virus discovery  Basic and applied genomic characterization  Examination of evolution  Identify key agents of veterinary or human health significance  Discussion of bat conservation biology significance

In addition, we intend to provide data for future studies of network analysis for key pathogens, bat species and potential spillover events as well.

- 19 -

Materials and Methods

Sample collection Sample collection was performed in several regions of Hungary from a total of 45 sampling locations between 2012 and 2014. Further sampling events were conducted in Georgia, Ukraine, Romania, Slovakia and Serbia, altogether in 16 sites, in 2014 (Fig. 3). It should be highlighted that all bat species in Europe are strictly protected under the Flora, Fauna, Habitat Guidelines of the European Union (92/43/EEC) and the Agreement on the Conservation of Populations of European Bats (http://www.eurobats.org). Invasive bat sampling is prohibited, hence we collected faecal droppings from bats that were captured primarily for bat-ringing or bat rehabilitation activities during the mating season of bats (end of August, beginning of September) at swarming sites and in their natural foraging habitats. All bat mist-netting and harp-trapping activities were undertaken using the basic recommendations outlined by Kunz et al. (2009). Animal handling processes were conducted by experienced chiropterologists with appropriate license for safe handling of bats, who identified captured animals for species according to Dietz and von Helversen (2004). Provider and number of licences are: Ministry of Energetics, Development, and Environmental Protection of the Republic of Serbia (353- 01-2660/2013-08); Hungarian National Inspectorate for Environment, Nature and Water (14/2138–7/2011); Ministry of Environment and Natural Resources Protection of Georgia (4752, 26 August 2014); Speleological Heritage Committee, Romania (33/2013, 97/2013, 418/2014, 119/2014).

- 20 -

Figure 3. Bat sampling was conducted between 2012 and 2014 in countries highlighted with dark grey colour. Sample numbers from these countries, involved in this study were as follows: Hungary (n=946), Georgia (n=9), Ukraine (n=25), Romania (n=63), Slovakia (n=5) and Serbia (n=79).

During mist-netting events, animals were freed from nets immediately and put into sterile, disposable, highly perforated paper bags individually and were left hanging for a maximum of 15 min to let them defecate (Fig. 4). After collecting faecal samples from the bags, bats were released at the netting sites. Duplicate sampling was prevented by marking captured individuals with paint. All samples were collected in 500 µL of phosphate buffered saline (PBS) and kept on dry ice until processed at the laboratory.

- 21 -

A

B

- 22 -

C Figure 4. Collection of bat faecal samples using mist-net (A), harp-trap (B). Individually sampling was solved using disposable paper bags (C).

Nucleic acid preparation Samples were homogenized with Minilys® personal homogenizer (Bertin Corp., USA) by adding one piece of 2.50-2.80 mm diameter glass beads (Kisker Biotech GmbH & CO., Germany) to each tube and homogenized for 30 sec at maximum speed. After homogenization, samples were centrifuged at 15,000 g for 10 min. Nucleic acid was extracted from 200 μl of supernatant using GeneJET Viral DNA/RNA Purification Kit (Thermo Fisher Scientific., USA) following the manufacturer’s recommendations. The extracted nucleic acid was eluted in 50 μl of elution buffer, and stored at -80°C until further laboratory processes.

Polymerase Chain Reaction (PCR) methods In order to detect viral-specific nucleic acid in our samples, various oligonucleotides were used, which are summarized in Table 1, indicating the type of PCR. All PCR workflows were previously optimized for the best annealing temperature. Reverse transcription polymerase chain reactions (RT-PCR) were performed with QIAGEN OneStep RT-PCR Kit (Qiagen, Germany), whilst longer fragments (>1000 bp) were obtained with SuperScript® III One-Step RT-PCR System with Platinum® Taq DNA Polymerase Kit (Thermo Fisher Scientific, USA). Conventional PCR reactions were done with GoTaq® G2 Flexi DNA Polymerase Kit (Promega, USA), long amplicons (>800 bp) were amplified with Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific, USA).

- 23 -

Table 1: Summary of oligonucleotide sets used to identify members of the virus families: Astroviridae (AstV), Caliciviridae (CalV), Coronaviridae (CoV), Filoviridae (FiV) Picornaviridae (PiV) and Circoviridae (CiV).

Oligonucleotides Final Type of PCR References amplicon size (nt)

AstV F1/1: 5´ -GARTTYGATTGGRCKCGKTAYGA- 3´ 422 Semi-nested RT-PCR Chu et al., F1/2: 5´ -GARTTYGATTGGRCKCGKTAYGA- 3´ 2008

R: 5´ -GGYTTKACCCACATNCCRAA- 3´

F2/1: 5´ -CGKTAYGATGGKACKATHCC- 3´

F2/2: 5´ -AGGTAYGATGGKACKATHCC- 3´

CalV p289: 5´ -TGACAATGTAATCATCACCATA- 3´ 319-331 RT-PCR Jiang et al., 1999 p290: 5´ -GATTACTCCAAGTGGGACTCCAC- 3´

CiV CVF1: 5´ -GIAYICCICAYYTICARGG- 3´ ~400 nested PCR Lima et al., 2014 CVR1: 5` -AWCCAICCRTARAARTCRTC- 3`

CVF2: 5´ -GGIAYICCICAYYTICARGGITT- 3´

CVR2: 5´ -TGYTGYTCRTAICCRTCCCACCA- 3´

CoV PC2S2: 5´ -TTATGGGTTGGGATTATC- 3´ ~440 nested RT- de Souza- PCR Luna et al., PC2As1: 5´ -TCATCACTCAGAATCATCA- 3´ 2007

PCS: 5´ - CTTATGGGTTGGGATTATCCTAAGTGTGA- 3´

PCNAs: 5´ - CACACAACACCTTCATCAGATAGAATCATCA- 3´

FiV FV-F1: 5’ – GCMTTYCCIAGYAAYATGATGG- 3’ 405 nested RT- Hu et al., PCR 2015 FV-R1: 5’ – GTDATRCAYTGRTTRTCHCCCAT- 3’

FV-F2: 5’ – TDCAYCARGCITCDTGGCAYC – 3’

FV-R2: 5’ – GIGCACADGADATRCWIGTCC – 3’

PiV PiVF: 5´ -CYT ATHTRAARGATGAGCTKAGA- 3´ ~400 RT-PCR Lau et al., 2011 PiVR: 5´ -GCAATNACRTCATCKCCRTA- 3´

Follow up PCR procedure primers along with sequence and strain-specific primer sets designed during this work are listed as a supplementary material.

- 24 -

Determination of the terminus of genomic RNA To obtain the appropriate sequence of the genome segment ends, a short oligonucleotide (PC3), phosphorylated at the 5’ end and blocked at the 3’ end with dideoxy cytosine, was ligated to the 3’ ends of the genomic RNA in the nucleic acid extract. Five µl total RNA was combined with 25 µl RNA ligation mixture (consisting of 3.5 µl nuclease free water, 2 µl of 20 µM PC3, 12.5 µl of 34% (w/v) polyethylene glycol 8000, 3 µl ATP, 3 µl 10X T4 RNA Ligase buffer and 10 U T4 RNA Ligase I (New England Biolabs, USA), then incubated at 17°C for 16 hours. Following the incubation, the RNA was extracted using the QIAquick Gel Extraction Kit (Qiagen, Germany). Binding of RNA to the silica-gel column was performed in the presence of 150 µl QG buffer from the extraction kit and 180 µl isopropanol. All subsequent steps were performed according to the manufacturer’s instructions. Five µl ligated RNA was heat-denatured in the presence of 1 µl of 20 µM primer (PC2, which is complementary to the PC3 oligonucleotide ligated to the 3’end) at 95°C for 5 min and then placed on ice slurry. The reverse transcription mixture contained 14 µl nuclease free water, 6 µl 5× First Strand Buffer, 1 µl of 10 µM dNTP mixture, 1 µl 0.1M dTT, 20 U RiboLock RNase Inhibitor (Thermo Scientific, USA) and 300 U SuperScript III Reverse Transcriptase (Invitrogen, USA). This mixture was added to the denatured ligated RNA and incubated at 25°C for 5 minutes and then 50°C for 60 minutes. The reaction was stopped at 70°C for 15 min. Subsequently, 2 µl cDNA was added to the PCR mixture, which consisted of 17 µl nuclease free water, 1 µl of 10 µM dNTP mixture, 2,5 µl 10× DreamTaq Green Buffer (including 20 mM MgCl2), and two µl of 20 µM primer pair (i.e. 1 µl PC2 and 1 µl gene-specific primer; data not shown) and 2,5 U DreamTaq DNA polymerase (Thermo Scientific, USA). The thermal profile consisted of the following steps: 95°C 3 min, 40 cycles of 95°C 30 sec, 42°C 30 sec, 72°C 2 min, and final elongation at 72°C for 8 min.

- 25 -

Cloning and Sanger Sequencing PCR amplicons were cloned into a pGEM-T Easy vector (Promega, USA), and Escherichia coli JM109–competent cells were transformed with the recombinant plasmid. Escherichia coli was incubated in Luria-Bertani medium (LB; Sigma Ltd.) supplemented with 100 mg/mL ampicillin as a selective agent. After incubation at 37 C for 12 h, positive clones were selected and the plasmids were extracted using a QIAprep Miniprep Kit (Qiagen, Germany). Target amplicons from the positive plasmids were amplified by standard PCR using pGEM-T Easy Vector-specific primers following the manufacturer’s instructions. Amplified DNA products were purified by the QIAquick Gel Extraction Kit (Qiagen, Germany) and prepared for sequencing using BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Biosystems, USA). Samples were sequenced bidirectionally on an ABI Prism 310 DNA Sequencer (Applied Biosystems, USA).

Next-Generation sequencing with Ion Torrent PGM Ion Torrent compatible libraries were prepared using the NEBNext® Fast DNA Fragmentation & Library Prep Set for Ion Torrent (New England Biolabs, USA) with the Ion Torrent Xpress barcode adapters (Life Technologies, USA). Emulsion PCR to obtain clonally amplified fragments was carried out using the Ion OneTouch™ 200 Template Kit (Life Technologies, USA) on a OneTouch version 2 equipment (Life Technologies, USA) as recommended by the manufacturer. Templated beads were enriched using an Ion OneTouch™ ES pipetting robot (Life Technologies, USA). The 200 bp sequencing protocol was performed on a 316 chip (Life Technologies, USA) using the Ion Torrent PGM (Life Technologies, USA) semiconductor sequencing equipment. Trimmed sequence reads were used for de novo assembly utilizing the MIRA (version 3.9.17) (Chevreux et al., 1999). Additional bioinformatic analyses, validation with remapping were performed using the CLC Genomics Workbench (version 6.5.1; http://www.clcbio.com) and the DNAStar (version 12; http://www.dnastar.com).

- 26 -

Recombination analyses In order to reveal potential recombination events, detected virus genomes were subjected to recombination analyses performed with SimPlot software version 3.5.1 (Lole et al., 1999).

Phylogenetic analyses Basic sequence manipulation and verification were performed using GeneDoc version 2.7 software. Nucleotide sequences were aligned by ClustalX version 2.0 software, and phylogenetic trees were constructed with MEGA version 6.0 software (Tamura et al., 2013). Automatic model selections were implemented by PhyML online version, Smart Model Selection (http://www.atgc-montpellier.fr/phyml-sms/) in order to use the best fitting phylogenetic model for each our datasets.

Protein modeling Protein model-based predictions were implemented only in case of Calicivirus characterisation. The 3D structure models of mature virus capsid structures were generated with Iterative Threading ASSEmbly Refinement (I-TASSER) server (Zhang, 2008; Roy et al., 2010). The models were built using the amino acid sequences of the newly identified viral strains. The following templates were used to thread the calicivirus viral capsid protein structures presented in this study: PDB ID codes 3M81 (Feline calicivirus (FCV) capsid protein), 2GH8 (X-ray structure of a native calicivirus) and Norwalk virus capsid (PDB ID code 1IHM). The raw protein model structures were refined with the MacroModel energy minimization module of the Schrödinger Suite (Schrödinger, 2015) to eliminate the steric conflicts between the side-chain atoms. Pairwise protein sequence alignments were calculated with the NeedleP tool of the SRS bioinformatics software package. The T=3 virion models were created with the Oligomer Generator application of VIPERdb (available at http://viperdb.scripps.edu/ oligomer_multi.php). Prior to virion model generations, the asymmetric units were constructed with Schrödinger Suite using the coordinates of subunit A, B and C of FCV and Norwalk virus in vdb convention format. Molecular graphics and sequence alignment visualization were prepared using VMD v1.9.1 (Humphrey et al., 1996) and the Multiple Sequence Viewer of the Schrödinger Suite, respectively.

- 27 -

Nucleotide composition analysis In order to predict potential host groups of the Circoviridae family members described in this study, nucleotide composition analysis (NCA) was performed. A set of 183 complete genome sequences were selected to be representative of Circoviridae family and were downloaded from the GenBank database. A list of the accession numbers for this control data set is provided in the supplemental material. Each sequence in the dataset was assigned to three host categories of mammalian, bird, and invertebrate. Sequences with unknown host were excluded from the analysis. To maximize discrimination between control sequences and infer the host origin of the sequences obtained in the current study, mononucleotide and dinucleotide frequencies for each sequence were determined using the ‘seqinR’ package (Charif & Lobry, 2007) for R 3.2.3 software (R Development Core Team, 2016) and used as canonical factors in the analysis. Discriminant analysis was performed using ‘candisc’ package for R (Friendly & Fox, 2016).

Pairwise identity calculation Pairwise identity calculations were implemented with Circoviridae family members only. Pairwise identity analysis was performed with Sequence Demarcation Tool software (SDT v 1.2) (Muhire et al., 2014).

- 28 -

Results and Discussion

Regarding the continuous sampling efforts during ongoing laboratory investigations, sample numbers involved in different sections may greatly vary. Altogether 1127 samples were involved in the thesis, representing all 32 bat species known from Europe with greatly varying numbers. Hungary was over-represented in our sampling efforts and experiments as well. Sample numbers involved in our studies per region were as follows: Hungary (n=946), Georgia (n=9), Ukraine (n=25), Romania (n=63), Slovakia (n=5) and Serbia (n=79). It should be noted that all further section may contain different starting materials, such as guano samples or tissue samples, which details are indicated separately in every section.

Astroviridae European large-scale surveillance studies were performed for the first time in Hungary, in order to reveal the diversity of bat-harboured astroviruses of multiple bat species (Kemenesi et al., 2014A; Kemenesi et al., 2014B) Altogether 447 specimens sampled in Hungary between 2012 and 2013, belonging to 24 species were tested for astroviruses, although the number of specimens from different bat species was variable (range, 1-125). Out of the 24 bat species tested, AstVs were identified in the following eight species: Miniopterus schreibersii, Myotis bechsteinii, Myotis daubentonii, Myotis emarginatus, Myotis nattereri, Nyctalus noctula, Pipistrellus pygmaeus, and Plecotus auritus. The overall detection rate of AstV in bats was 6.93% (95% C.I.: 4.854, 9.571), varied significantly between species (2.7 - 80%), with M. schreibersii showing significantly higher rates than any other species (p < 0.001). AstVs were detected in 16 out of the 45 collection sites in Hungary. Upon sequence and phylogenetic analysis of a fragment of the RNA-dependent RNA-polymerase (RdRp) gene, the novel Hungarian bat AstV (BtAstV) strains (GenBank Accession: KJ652321–KJ652328) clustered with other BtAstV strains identified worldwide, and markedly differed from other mammalian AstVs (Fig. 5). In agreement with previous studies, we also observed a notable genetic variability within the BtAstV strains. Genetically diverse virus sequences were determined from the Myotis spp. (M. daubentonii, M. nattereri, M. emarginatus, and M. bechsteinii), Miniopterus spp., Pipistrellus spp., Plecotus spp., and Nyctalus spp., with patterns of segregation apparently related to the various bat species.

- 29 -

Nucleotide identity between the novel BtAstV strains detected in Hungary and other BtAstVs detected worldwide ranged from 51% to 75%. However, the average nucleotide divergence between AstV species recognized by the International Committee on Taxonomy of Viruses was calculated as 55%. On the basis of the low genetic divergence in the RdRp gene, the novel AstV strains identified in the Hungarian bats might represent potentially new species of AstVs (Fig. 5). The first report on BtAstVs was published in 2008 (Chu et al., 2008). Since then, only a few additional studies revealed bats as reservoirs of AstVs in Europe and Asia (Zhu et al., 2009; Drexler et al., 2011; Anthony et al., 2013). Only M. myotis, M. daubentonii, M. bechsteinii, and P. auritus have been addressed before as AstV reservoirs in Europe. During our study, we described five new bat species as potential reservoirs for AstVs (Kemenesi et al., 2014). Most of BtAstV positive samples detected in our study originated from Schreiber's Bent-winged Bat (M. schreibersii) from a single colony with an outstanding prevalence of ~80%. Schreiber’s bat is the only bat species in Hungary that roosts almost exclusively in underground shelters (Gombkötő et al., 2007). These colonies are usually large and dense because they can save considerable amount of energy if their bodies are in close contact during the hibernation period. These bats may roost together with R. ferrumequinum, R. euryale, M. myotis, Myotis blythii, and M. emarginatus. Miniopterus schreibersii is one of the fastest flying bats in Europe and can travel large distances (>500 km) from one roost to another (Hutterer et al., 2005; Hutson et al., 2008). All of these factors may contribute to the higher detection rate of AstVs in this species, but because all samples originate from only a few locations, we cannot rule out that only these populations have such a high infection rate. The hypothesis that large colonies as in case of M. schreibersii favor the spread of different viruses (i.e., AstVs), is supported also by previous studies in which marked variations were observed in the rates of detection, varying between 0.8% and 36.4%, depending on the geographic area and colony examined (Xiao et al., 2011).

- 30 -

Figure 5. Phylogenetic analyses of novel astroviruses (BtAstV). The phylogenetic tree was constructed based on a 420 bp region of the RNA-dependent RNA polymerase gene. The Hungarian BtAstV strains detected in this study are marked in boldface.

Caliciviridae Altogether 447 specimens sampled in Hungary, belonging to 24 species were tested for caliciviruses, although the number of specimens from different bat species was variable (1- 125). Novel strains of bat caliciviruses (BtCalV) were detected in three bat species M. daubentonii, Myotis alcathoe, Eptesicus serotinus. The detection rate of BtCalV among the sampled bats was 0.67% (95% C.I.: 0.189, 1.780). A sequence similarity search using BlastN against the National Center for Biotechnology Information (NCBI) nonredundant nucleotide database characterized the viruses as members of the Caliciviridae family. Which observation was further confirmed via phylogenetic analyses, based on a partial 320 nt segment of the RNA- dependent RNA polymerase gene (Fig 6).

- 31 -

Figure 6. Unrooted maximum likelihood tree of novel bat caliciviruses (BtCalV) on the basis of a partial (320 nucleotide) sequence of the RNA-dependent RNA polymerase gene. Novel BtCalV strains are marked in boldface.

Strain BtCalV/M63/HUN/2013 (GenBank Accession: KJ652319), detected from M. daubentonii, was more closely related to porcine enteric sapoviruses, differing from bat sapoviruses identified in China, suggesting this strain may be a member of the Sapovirus genus. Strain BtCalV/BS58/ HUN/2013 (GenBank Accession: KJ652318), identified from E. serotinus, appeared as an outlier between the genera Recovirus and Valovirus. Even more interesting, strain BtCalV/EP38/HUN/2013 (GenBank Accession: KJ652320) identified from M. alcathoe, segregated with avian CalV strains, rather than with other mammalian viruses.

- 32 -

In order to characterize more in detail the bat caliciviruses, large portions of the genome sequence of BtCalV/M63/HUN/2013 and BtCalV/BS58/ HUN/2013 were successfully determined by 3’ RACE PCR method. Unfortunately multiple efforts of 3’ RACE PCR were unavailing in case of BtCalV/EP38/HUN/2013 strain. Since 5’ RACE PCR protocols were not successful for the examined samples, we obtained a 3278 and 3130 nt long partial genome sequence for the strains BtCalV/M63/HUN/2013 and BtCalV/BS58/HUN/2013 respectively. The sequenced genomic fragment spanned the partial RNA-dependent RNA polymerase (RdRp) gene (M63, 879 nt; BS58, 841 nt) and the whole coat protein coding region (1545 nt and 1436 nt) along with the complete VP2 protein coding region (656 nt and 668 nt). The calicivirus conserved aa motifs GLPSG and YGDD (Smiley et al., 2002) were identified in the RdRp region. A third calicivirus RdRp motif DYXXWDST, was also well conserved in both viruses, exhibiting the following aa sequence DFSKWDST (Wolf et al., 2011). Based of phylogenetic analysis of larger genomic fragments, strain BtCalV/M63/HUN/2013, detected in a M. daubentonii bat, was grouped with other bat-derived sapoviruses and showed the closest relationship with a Chinese sequence (GenBank Accession: KJ641703), detected from the faeces of a Myotis myotis bat (Fig. 7). The similarity between BtCalV/M63/HUN/2013 and KJ641703 resulted in 67% nt and 67% aa identity in the RdRp and 66% nt and 61% aa identity in the capsid gene. Strain BtCalV/BS58/HUN/2013, derived from an E. serotinus bat species, was grouped with a bat calicivirus and with porcine caliciviruses of the proposed genus Valovirus. The closest phylogenetic relationship was described with a Chinese bat calicivirus strain, BtMspp.-CalV/GD2012 (GenBank Accession: KJ641700), detected in an undefined Myotis species. Identity was 63 nt and 60% aa in the RdRp and 63% nt and 56% aa in the capsid gene between strain BS58/HUN/2013 and strain BtMspp.-CalV/GD2012 (Fig. 7).

- 33 -

Figure 7. Phylogenetic tree of the two novel bat calicivirus sequences and their relationship with other known genera within the family Caliciviridae identified from human, swine and bats. Phylogenetic trees were constructed based on a partial sequence of 3130 nucleotides, incorporating the whole capsid region of the viruses. Strains reported in this study are marked with black dots, and all bat derived calicivirus strains are marked with a grey background. The phylogenetic trees were constructed with MEGA v5.0 software using the Maximum-Likelihood method, based on the General Time Reversible model with Gamma Distribution (GTR+G). Number of bootstraps for simulations was 1000.

- 34 -

This geographically distant evolutionary relationship among European and Asian originated bat viruses has also been discussed in other chapters of this thesis. These new results, with related caliciviruses in Myotis and Eptesicus bats, widens the list of bat species harbouring genetically related viruses in large geographic distances, which can be a result of an ancient evolutionary history of bats and their viruses and presumably could be an outcome of the biogeographical outcome of laurasiatherian origins of bats. In-depth structural analysis of the capsid of caliciviruses was performed as well. Homology modelling allowed a more precise characterization of the two novel bat caliciviruses discussed before. There was 27.3% aa identity, and 42.3% aa similarity between the template FCV capsid protein (PDB ID: 3M8L) and the mature CP of the bat calicivirus strain BtCalV/M63/HUN/2013. Likewise, there was 34.7% aa identity and 47.6% similarity, between the template Norwalk virus CP (PDB ID: 1IHM) and the CP of the bat calicivirus strain BtCalV/BS58/HUN/2013 (Fig. 8a-b). These identity and similarity values allowed reliable homology modelling and reconstruction of the capsid and virion structure of the two viruses. The mature calicivirus capsid protein has three main domains: S, P1 and P2 (Fig. 8c-d). The S (i.e. shell) domain is composed of a canonical eight-stranded β-barrel structure. This domain forms the inner part of the virion and it has the most conserved structure within caliciviruses. The P1 and P2 subdomains build up the middle and the outer protruding part of the virion, respectively. The hypervariable regions, the primary targets of neutralizing antibodies, are located on the P2 subdomain (Ossiboff et al., 2010). The basic structure together with the recognized main domains was predicted to be conserved in the two Hungarian bat caliciviruses. However, in the P2 region, we detected three deletions and an insertion (Fig. 8a-c) in the CP of the bat calicivirus BtCalV/M63/HUN/2013 when compared to the FCV template sequence. In the case of the strain BtCalV/BS58/HUN/2013, we found two insertions and two deletions on the P2 subdomain (Fig. 8 b-d) with respect to the template structure. Insertions and/or deletions in the P2 domain are common even among caliciviruses of the same genus and/or genotype (Pinto et al., 2012; Medici et al., 2015). These small structural and sequential differences may play important roles in the P2 subdomain dual functions. The protruding regions of the virion may be involved as antigens in the humoral immune response (Fig. 8 e-f). Furthermore, this variable part of the P2 subdomain may take part in protein-protein interactions between the CP and its functional cell receptor. Additionally, these regions are responsible for the fine tuning of cell-virus interaction during cell entry (Ossiboff et al., 2010).

- 35 -

In summary, genome sequencing, phylogenetic analysis and homology modelling allowed a more precise characterization of these novel bat caliciviruses. BtCalV/M63/HUN/2013 could be firmly assigned to the Sapovirus genus. Sapoviruses are enteric viruses associated with gastroenteritis in humans and with enteritis in pigs (Oka et al, 2015). These viruses have been also identified in diarrheic dogs and cats (Soma et al., 2015; Li et al., 2011B) and display an impressive genetic heterogeneity (Oka et al., 2015). Interestingly, genetic heterogeneity is also displayed by the few bat sapoviruses identified thus far, thus posing a challenge for the design of specific diagnostic tools. Based on our phylogenetic analyses species-specific patterns are clear among sapoviruses of different animal origin (Fig. 8). However, high phylogenetic diversity can be observed within these species- related clusters with several separate branches, which indicates a long-term evolutionary diversification. On the other hand, BtCalV/BS58/ HUN/2013 was genetically more similar to porcine caliciviruses of the putative novel genus Valovirus. The genomic organization of the bat calicivirus was not identical to the organization observed in valoviruses. The closest phylogenetic relatedness was observed with valoviruses, however, the novel bat calicivirus sequence clearly formed a separate branch. The limited number of studies describing bat caliciviruses is providing evidence that these viruses are highly heterogeneous genetically. It is possible that as data will accumulate, this picture will be confirmed and the interactions between caliciviruses from bats and from other animal species, including humans, will be more evident. Also, this will help clarify whether caliciviruses may have a pathogenic role in bats and will help reveal the evolutionary pathways of these viruses across the various bat species and geographic areas.

- 36 -

- 37 -

Figure 8. Structure comparison of mature calicivirus (ORF2, VP1 segment) CP proteins and virions. Structure-based amino acid sequence alignments of the newly reported bat calicivirus capsid proteins and the template FCV and Norwalk calicivirus VP1 proteins (a) and (b). The background of the sequence alignments reflects the homology levels of the two-two related capsid protein sequences: identical amino acids are red, similar aas. are light orange while different aas. are light pink. The main structural differences are indicated by shades of magenta and green colour codes on the sequence alignment and on the superimposed CP structures. The template calicivirus VP1 protein structures are illustrated in cyan cartoon representation, while the new bat calicivirus VP1 model structure are pink (c) and (d). Molecular surface representation of the superimposed template and the newly described bat calicivirus virions (e) and (f). The molecular surface is coloured by radial extension of the amino acids from the virion centre. Dark blue represents the most protruding CP parts. The structural differences were coloured in the same way as for the capsid monomers.

- 38 -

Carmotetraviridae We used viral metagenomics in order to investigate Western barbastelle bat (Barbastella barbastellus) guano samples, collected at various sampling sites in Hungary, during 2014. A total of 15 guano samples were collected in the sampling year of 2014, all were subjected to IonTorrent PGM sequencing. Collection sites are distributed in multiple regions of the country: Bakony mt., Bükk mt., Mecsek mt. Interestingly, a novel Providence virus strain (14EPF155) was detected from a female B. barbastellus collected in Nagyvisnyó, Hungary, in September 2014, which was only known from the new world to date (Walter et al., 2010). The genomic organization of the Hungarian PrV strain corresponded to the strain vFLM1, previously isolated in the United States (Walter et al., 2010). The phylogenetic position of this strain was monophyletic with the vFLM1 strain, and the Hungarian variant unambiguously clustered within the Carmotetraviridae family (data not shown). The complete genome of 14EPF155 was 6,158 bp long. All major ORFs were detected (viral replicase p40/p140 with a 413/970-amino acid (aa) length; p81 capsid protein precursor with 754 aa; and p130 nonstructural protein of unknown function with 1,221 aa) along with the read-through stop codon at the termini of p40 replicase. Nucleotide sequence identity compared to the vFLM1 strain was 85% regarding the entire genome, while aa identity was 76% for p130; 91% for p104; 86% for p40; and 87% for the p81 capsid precursor gene. Dietary viruses from bat guano samples were previously described as constituting the majority of viral sequence reads (Li et al., 2010; Wang et al., 2015), although the original host of these viruses is unpredictable without further examination. A close relative of its original host, namely, the Old World cotton bollworm Helicoverpa armigera, which is a significant pest of agriculture in Asia, Europe, Africa, and Australia (Tay et al., 2013), may serve as a host. Further studies will be required to decipher the true host of the Hungarian PrV strain. Interestingly, Providence virus was recently described as a possible human-infecting virus on HeLa cell line in vitro (Jiwaji et al., 2016). This widens the possible host spectrum of the virus, which now includes mammals, and raises the question about the possible effect on bat colony health status, and its’ human health relevance as well.

- 39 -

Coronaviridae Altogether 447 specimens sampled in Hungary, belonging to 24 species were tested for coronaviruses (CoVs). CoV-related RNA was detected in seven bat species: Myotis daubentonii, Myotis myotis, Myotis nattereri, Pipistrellus pygmaeus, Rhinolophus euryale, Rhinolophus ferrumequinum, and Rhinolophus hipposideros. The overall detection rate of bat coronaviruses (BtCoV) among the sampled bats was 1.79% (95% C.I.: 0.849, 3.348). Bats were found positive for coronaviruses in seven sampling locations. BtCoV was identified in three European bat genera and seven species. SARS-like CoV (GenBank Accession: KJ652335) was detected in the species R. euryale, while alphacoronavirus sequences were obtained from R. ferrumequinum, R. hipposideros, M. daubentonii, M. myotis, M. nattereri, and P. pygmaeus bats (GenBank Accessions: KJ652329–KJ652334). According to the RdRp gene-based phylogenetic analyses (Fig. 9), the novel Hungarian BtCoV strains clustered together with other previously reported BtCoV strains from Germany and Bulgaria. The Hungarian BtCoV strains of the alphacoronavirus group displayed 52-96% nucleotide identity to non-Hungarian alphacoronaviruses, whereas the Hungarian betacoronavirus strains displayed 82-96% nucleotide identity to other betacoronaviruses.

Figure 9. Phylogenetic tree of bat-transmitted alpha- and betacoronaviruses (BtCoV) detected in Hungary. Analysis was performed based on a 440-nucleotide segment of the RNA-dependent RNA polymerase gene. BtCoV strains identified in this study are marked in boldface.

- 40 -

Many bat species are proved to be natural reservoirs for a variety of CoVs worldwide, including SARS- and MERS-related coronaviruses (Brandao et al., 2008; Corman et al., 2013; Ithete et al., 2013; Góes et al., 2013; Annan et al., 2013; Yang et al., 2014; Ge et al., 2013). In recent years, a wide range of CoVs has been detected among European bat species including the United Kingdom, Germany, the Netherlands, Bulgaria, Slovenia, Hungary and France. The overall detection rate of coronaviruses in our study (1.79%) was considerably lower than the values reported in these European countries (Gloza-Rausch et al., 2008; Drexler et al., 2010; Reusken et al., 2010; Rihtaric et al., 2010; August et al., 2012; Annan et al., 2013; Goffard et al., 2015). The higher detection rates in previous European studies are mainly due to their high detection rates among Pond bats (Myotis dasycneme). In Hungary, we found no evidence for raised detection rate in specimens originating from M. dasycneme compared to other species, although we tested only 11 individuals. Additional investigations are needed to solve this discrepancy, involving a greater number of samples collected from M. dasycneme. The growing urbanization of these species may result in a greater frequency of interactions between humans and bats. The three sets of CoV-specific primers used in our study showed great differences in detection success rates. We found that primers published by de Souza-Luna et al. (2007) were the most appropriate for a primary surveillance of bat CoVs from faecal samples. This might be explained by the high genomic diversity of CoVs and the different specificity of the primer sets even within the highly conserved region targeted by the primers. Because bats may harbour divergent CoVs highly pathogenic to humans and/or domestic animals (such as SARS and MERS coronaviruses), the systematic comparison and further improvements of various diagnostic assays that are suitable to detect potential zoonotic CoVs seem crucial from both public health and veterinary perspectives, as described previously by Memish et al. (2013).

- 41 -

Picornaviridae Thirteen faecal samples were collected from a single M. schreibersii bat colony, at a cave in Szársomlyó mountain, Hungary (GPS coordinates 45°51′17 N; 18°24′40 E), in 2013. In the sampling year the bats had an estimated nursery colony size of 1000 individuals, and 1500 individuals for transient colony size. In two samples, BatPV sequences were obtained by metagenomic analyses with 416 bp and 465 bp consensus length represented by 14 and 15 sequence reads, respectively. Another four stool samples collected from animals in the same colony were found to contain BatPV RNA when tested by specific primer sets (Supplementary Material). The genomic RNA sequences identified by viral metagenomics were used to design virus-specific primers which permitted the primer-walking sequencing and the 5′/3′ RACE. As a result, the near-complete viral genome (7947 nt) was sequenced for six BatPVs. Using the reference BatPV strain (GenBank Accession: JQ814851), we estimate that approximately 500 nt of sequence data were missing at the 5′ UTR. However, the complete coding region was determined and analysed in detail. We identified a single ORF that encoded a large polyprotein of 2284 aa (Figure 10.).

Figure 10. Genomic organization of the newly described picornavirus strain. Conserved amino-acid motifs, gene names and regions along with main polyproteins are indicated.

Sequences of the novel BatPV were deposited in GenBank (Accession: KP054273– KP054278). The new BatPV showed the characteristic genome organization of 5′ UTR[L- P1(VP0, VP3, VP1)-P2(2A, 2B, 2C hel)-P3(3A, 3B VPg, 3C pro, 3D pol)]3′ UTR-poly(A). The amino acid lengths of different genome regions were 97 (L), 810 (P1), 576 (P2) and 801 (P3), respectively. The L protein did not possess either the catalytic dyad (Cys and His) of papain- like thiol proteases (Gorbalenya et al., 1991) or a CHCC zinc-binding motif (Chen et al., 1995).

- 42 -

With the exception of cleavage sites VP0/VP3 and VP3/VP1, the same cleavage pattern was observed between the Hungarian BatPV strains and the prototype reference strain identified previously by Wu et al. (2012) (Figure 10.). Using the conserved domain database (CDD) search (Marchler-Bauer et al., 2011), Rhv-like capsid domains (cd00205), 2C RNA helicase (pfam00910), 3C cysteine protease (pfam00548) and 3D RdRp (cd01699) characteristic picornaviral motifs were recognized. Amino-acid motifs are highlighted and summarized on Figure 10. Based upon sequence data and secondary structural similarities, the Hungarian BatPV was predicted to possess a type II IRES (Fig. 11) similar to those of foot-and-mouth disease virus (FMDV), encephalomyocarditis virus (EMCV) and cosaviruses (CoSV). Interestingly, bat-transmitted picornaviruses that were classified into the Sapelovirus genus possess a type IV IRES (Lau et al., 2011).

- 43 -

Figure 11. (a) Predicted secondary structure for 5′ UTR of the novel BatPV strain (BatPV- V13/13/Hun). Stem-loops are labelled according to Kapoor et al. (2008) and Duke et al. (1992). Bases that are identical or highly conserved between BatPV, EMCV and CoSV are indicated by grey shading. Initial codon is underlined. Py, polypyrimidine tract. (b) Predicted secondary structure for 3′ UTR of the novel BatPV. Grey boxes indicate the conserved nucleotide motif identified previously in EMCV (Duque & Palmenberg, 2001).

- 44 -

The main conserved elements, H, I, J, K and L, of the type II IRES were identified in the 5′ UTR. Particularly, the predicted secondary structure included a series of signature elements characteristic of type II IRES, e.g. the GNRA tetranucleotide and two A/C-rich loop regions at the apical parts of the I domain, as well as the A-rich loop at the junction of the J and K domains. Domain F within the 5′ UTR of BatPV contained a sequence of 13 nt (AGGGUUCUAUCCU), identical to that found in CoSV, while domain H contained 9 nts (GGUCUUUCC), identical to EMCV. There were also high nucleotide similarities in the case of domains D and J between BatPV, CoSV, FMDV and EMCV. Additionally, BatPV also contained an 8 nt oligopyrimidine tract immediately downstream of the last stem-loop, L (Kapoor et al., 2008; Duke et al., 1992). Interestingly, the region from positions 329 to 440 showed both sequence and structural similarities to that of CoSV, consisting of putative stem– loops D, F and G, and a 20 nt polypyrimidine tract (positions 364–383). The 3′ UTR (Fig. 11) showed 76 % identity to the prototype Mischivirus A (Wu et al., 2012) and was determined to be 211 nt (including the stop codon). In addition, the 3′ UTR shared 88 % identity to Theiler's murine encephalomyelitis viruses (TMEVs) (Buckwalter et al., 2011; Law & Brown, 1990; Liang et al., 2008; Myoung et al., 2007; Pevear et al., 1987; Sorgeloos et al., 2013) between nt 71 and nt 110. Using Mfold (Zuker, 2003), the secondary structure of the new BatPV 3′ UTR was predicted to consist of six domains (A–F). Domain C contained the loop AAGCCA

88–93 motif present in domain I of EMCV, while domain D contained a variant sequence

(CCUUCU 115-120) that resembled the UCUUCU motif of EMCV (Duque & Palmenberg, 2001). The 3′ UTRs of the known picornaviruses contain stem–loops of variable size and number, which may be specific binding sites of viral or cellular proteins (Rohll et al., 1995). The complexity of the Hungarian BatPV 3′ UTR suggested the same role in virus replication; however, this hypothesis remains elusive until a cell-culture system for BatPVs becomes available.

Phylogenetic analyses (Fig. 12) of the entire coding region showed that the new BatPVs belong to the Mischivirus genus and they are most closely related to Mischivirus A, the only described member of the genus detected in M. schreibersii bats from China (Wu et al., 2012). The complete P1 (2429 nt, 810 aa), P2 (1727 nt, 576 aa) and P3 (2402 nt, 801 aa) regions show 32–37 %, 35–39 % and 29–32 % amino acid identity to other bat-derived picornaviruses from the Sapelovirus genus. On the other hand, amino acid identities between the Hungarian BatPVs and Mischivirus A were 73 %, 73–74 % and 67 %, respectively.

- 45 -

The main amino acid motifs found in the reference Mischivirus A species have also been described in our BatPV strains. When compared to the reference Mischivirus A species, minor differences were found in the length of the L protein (6 aa shorter), the P2 polyprotein (1 aa longer) and the P3 polyprotein (3 aa shorter).

Figure 12. Phylogenetic analyses of the novel BatPV strains detected in Hungary. Phylogenetic trees were reconstructed based on nucleic acid sequences of a 6852 bp coding region of the genome. Bat-derived picornaviruses, identified in previous studies, are indicated in grey. Hungarian BatPV strains identified in this study are marked in bold face.

- 46 -

We report the first molecular data and genomic characterization of a novel picornavirus from the bat species, M. schreibersii in Europe. Based on the phylogenetic analyses, the novel BatPVs (BatPVs) unambiguously belonged to the Mischivirus genus and was highly divergent from other BatPVs of the Sapelovirus genus. Although the Hungarian viruses were most closely related to Mischivirus A, they formed a separate monophyletic branch within the genus. Genome organization of the new BatPVs (BatPVs), along with the hypothetical cleavage sites and characteristic amino acid motifs were also similar to the prototype Mischivirus A. It remains to be clarified whether the occurrence of mischiviruses follows a particular geographical pattern and can be linked to different lineages of the widespread M. schreibersii bat species suggestive of co-evolution and/or co-adaptation. Our results suggest Mischiviruses and bats as a remarkable example of virus-host co-evolution which needs to be examined in the future involving more virus strains from different locations across Africa and Eurasia (the distribution range of Miniopteridae bats).

- 47 -

Reoviridae A total of 128 Miniopterus schreibersii bats were captured (45 males and 83 females) on October 3rd 2014 at cave Pionirska pećina (Beljanica Mt., Serbia; 44.07289° N, 21.64055° E). Fecal samples were collected from ten specimens (3 males and 7 females). Droppings were stored in RNAlater RNA Stabilization Reagent (QIAGEN, Germany) and kept on ice until laboratory processing. To explore the viral diversity six faecal specimens collected form Schreiber’s long-fingered bats were processed for viral metagenomics. In these samples, various amount of sequence reads mapped onto known eukaryotic viral sequences (range, <<0.1% to 0.9%). In one sample Reovirus (RV) sequences were the most abundant genomic traits (98.5%); however, these sequence reads were distributed among various RV species (incl. RVB, RVG, RVH and RVI). The library DNA that contained the most abundant RV-specific reads was resequenced. The resulting >1.3 Million sequence reads were subjected to de novo assembly. As a result, the consensus genome sequence of strain BO4351/Ms/2014 could be assembled from a total of 36,630 sequence reads at 131 X (segment 3) to 457 X (segment 11) average coverage. The assembled genome of RVJ was 18,135 bp in length (range, 3533 bp for segment 1 and 620 bp for segment 11). Terminal sequences at the 5’ ends showed relatively conserved structure with stable nucleotides at positions 1, 2 and 4 and some variations at positions 3, 5, and 6 (segments 1 and 2, GGCACA; segments 3 and 4, GGCATT; segments 5, 7 and 9, GGAAAT; segments 6 and 10, GGCAAA), while at the 3’ ends the variation was smaller (TAT/CACCC). Each segment had non-translating regions at both 5’ end (length range, 6 to 57 nt) and 3’ end (length range, 20 to 84 nt). Encoded proteins were assigned based on significant hits through the Blast engine and conserved peptide motifs. With this approach, we found the equivalents of the major structural (i.e. VP1 to VP4, VP6 and VP7) and non-structural (NSP1 to NSP5) proteins. Additional putative ORFs were predicted to be encoded on segments encoding VP6 and NSP5; however, these putative proteins shared no conserved protein motifs with those known from other rotavirus species. In the phylogenetic analyses cognate sequences of representative RVA to RVH strains (except RVE, for which no sequence information is available) were included (Fig. 13). The novel bat-harboured RV consistently clustered with clade 2 RV strains, and in particular, with porcine and human RVH strains. One exception was found when analyzing the NSP4 tree, where the limited support at the deepest nodes prevented the separation of the two major RV clades (Fig. 14).

- 48 -

Consistent with the phylogenetic analyses the greatest nucleotide and amino acid sequence identities for the novel bat-related RV were seen when compared to reference RVH strains (range, 14 (nt%) and 35 (aa%) for NSP4; 63 (nt%) and 64 (aa%) for VP1) (Table 2).

Table 2: Percentile nucleotide (nt) and amino acid (aa) sequence based identities between the novel bat- borne RV strain, BO4351/Ms/2014, and reference RVA-RVD and RVF-RVI strains. The Muscle algorithm within the TranslatorX online platform (Abascal et al., 2010) was used to obtain codon-based multiple alignments. Nucleotide and deduced amino acid sequence alignments were visualized in GeneDoc (Nicholas et al., 1997), whereas sequence distances were calculated with the MEGA6 program (Tamura et al., 2013) using the p-distance algorithm. Results obtained by this method were used to calculate sequence identity values.

RVA RVB RVC RVD RVF RVG RVH RVI Proteins nt aa nt aa nt aa nt aa nt aa nt aa nt aa nt aa VP1 40 25 58 57 41 24 41 25 42 24 60 59 63 64 61 59 VP2 34 14 54 46 35 14 34 12 36 14 56 47 62 61 54 45 VP3 38 17 46 32 38 19 36 16 38 18 46 31 56 49 51 36 VP4 33 11 41 25 35 14 35 12 35 12 42 25 48 34 44 26 VP6 35 15 50 39 34 17 34 13 32 11 49 39 55 49 52 46 VP7 38 16 42 22 36 16 36 14 37 16 43 25 52 37 49 29 NSP1 32 <10 39 21 30 <10 31 <10 31 <10 39 18 47 34 44 26 NSP2 38 16 56 48 35 17 38 17 38 17 55 47 63 59 56 46 NSP3 36 15 44 27 34 11 38 18 33 11 40 26 56 49 40 22 NSP4 34 10 36 15 32 12 34 12 33 12 36 10 41 14 35 13 NSP5 38 13 43 28 38 13 34 11 32 11 47 25 50 39 46 31

To place the novel bat-related strain, BO4351/Ms/2014, into the latest RV taxonomic framework, additional VP6 gene sequences were selected from GenBank to represent a broader genetic diversity of various RV species. In this analysis, again, BO4351/Ms/2014 was most closely related to the major genetic lineage containing RVH strains (49-50%, aa) and showed lower similarity to other clade 2 RVs (RVB, 39%, RVG, 39%). The genetic relationship of BO4351/Ms/2014 to clade 1 RVs was marginal (max. identity with RVC, 17%) (Table 2). Thus, applying the official species demarcation sequence cut-off value, which is 53% identity at the amino acid level, we conclude that the novel bat-harboured RV strain may represent a new RV species, tentatively called Rotavirus J (RVJ). The reference strain was therefore assigned as RVJ/Bat-wt/SRB/BO4351/Ms/2014/G1P1. To determine whether RVJ was common among the Schreiber’s bat in the cave under investigation, a nested PCR assay was developed targeting a sequence region that is conserved within the VP6 coding gene of both RVH and RVJ. By adapting the nested PCR assay that amplified a 338 bp long fragment (Supplemetary Material), another four stool samples were

- 49 - found to be positive for RVJ. Notably, given that RVs have been detected exclusively in birds and mammals, the dietary origin of the novel RV could be excluded at great confidence. Thus, the data presented here suggest that bats may be a true host species of RVJ.

Figure 13. Phylogenetic analysis of the VP6 gene. A total of 258 representative sequences were selected to provide a more comprehensive phylogenetic analysis of the VP6 gene. Color codes are indicated below the tree. Similarity plot prepared from amino acid sequences of the VP6 protein. The dashed line indicates the rotavirus species demarcation sequence identity cut-off value determined by Matthijnssens et al. (2012).

- 50 -

Figure 14. Phylogenetic trees obtained for the genes encoding all major structural proteins (VP1 to VP4, VP6, and VP7) and non-structural proteins (NSP1 to NSP5) with representative strains of RVA to RVI. Alignments were created using the TranslatorX online platform (http://translatorx.co.uk/). Phylogenetic trees were prepared using the neighbour-joining method as implemented in Mega6 (http://www.megasoftware.net/). Bootstrap values are shown at the branch nodes.

- 51 -

Recent years have witnessed considerable sequence data accumulation in public databases pointing out the enormous genetic diversity within the Rotavirus genus. Viral metagenomics largely contributed to our understanding of this genetic diversity. Until the early 2000s, RVA to RVG were considered as the only extant RV species (Estes et al., 2007). Sequence-independent amplification followed by cloning and sequencing led to the discovery of a novel human RV species and later, together with closely related porcine and bat origin strains, all classified into RVH (Hull et al., 2016; Nyaga et al., 2016; Kim et al., 2016). During our investigations, we described a novel RV detected in Serbian Schreiber’s long-fingered bats. This novel bat-related RV belongs to clade 2 RVs, which also includes RVB, RVG, RVH and RVI. Of interest, the novel strain was closely related to representative strains of RVH suggesting that these RVs had diverged from a common ancestor. However, molecular classification of the Serbian bat-related RV strain indicated that the Serbian bat RV strain is the first member of a novel RV species, RVJ. With the advent of sequence data, it will be possible to explore the geographic spread and genetic diversity within this candidate new RV species. Collaborative investigations among researchers involved in bat-related virus surveillance may help collect the missing information about host range and geographic dispersal of RVJ. Moreover, many bat-harboured viruses are capable of infecting and causing severe disease in human beings (Kohl & Kurth, 2014). Thus, it will be important to study whether the novel RVJ strains pose any occupational risk for professional chiropterologists or individuals contacting bats and their excreta.

- 52 -

Parvoviridae Parvovirus-related sequence data were obtained via metagenomics approach from faecal samples collected from a single Miniopterus schreibersii bat colony, at a cave in Szársomlyó mountain, Hungary (GPS coordinates 45°51′17N; 18°24′40E), 2013. These consensus sequences were 250 and 127 nt long and were represented by 24 and 2 sequence reads, respectively. We designed two sets of primers for further genome amplification efforts. PCR screening was implemented with BParV-F and BParV-R primers (Supplemetary Material) on the remaining M. schreibersii samples from the sampling site. As a result, 5 out of the total 13 samples were found to be positive. Based on the two consensus sequences, another set of primers (BParV Long-F and BParV Long-R – Supplementary Material) were designed and used in order to obtain larger genomic fragment. A 3438 nt long fragment of the genome (encompassing a 975 nt of the NS1 region and a 2013 nt segment of the VP1/VP2 genes) was obtained via long-range PCR and used further in genetic analyses. In addition to the NS1 and VP1/VP2 regions, a smaller ORF, encoding the putative middle protein (PMP) was also identified with a length of 450 nucleotides. Phospholipase A2 motif of the VP1 region was identified, which is typically present in most of the parvoviruses, but absent in other viral families. A Walker loop motif (GXXXXGK T/S) was identified in the NS1 region as GPASTGKS in our sequences (Fig. 15), same as described previously in human bufaviruses (Phan et al., 2012; Yahiro et al., 2014). The nucleotide sequence identities with the reference Chinese bat parvovirus strains fell between 77% and 81%. On the other hand, nucleotide sequence similarity with viruses in the Parvovirinae family ranged between 45% and 47%, while the amino acid sequence identity ranged between 35% and 42%.

Based on the phylogenetic analyses (Fig. 16), the novel BtBVs (GenBank Accessions: KR078343-“BtBV/V3/HUN/2013” and KR078344-“BtBV/V7/HUN/2013”) were closely related to human bufaviruses belonging to the Protoparvovirus genus and Primate protoparvovirus 1 species. Hungarian BtBV sequences were highly similar to those viruses detected previously from bats in China (unpublished data, GenBank Accessions: KC154061 and KC154060), which therefore are also related to bufavirus sequences. Although the Hungarian viruses were closely related to bufaviruses, they formed a separate monophyletic branch. Phylogeny revealed that bat-related bufaviruses might have had common unknown ancestors with bufaviruses identified from humans so far. In a recent publication, a novel parvovirus (WUHARV) was described in nonhuman primates, which were most closely related to bufavirus 2 genotype (Handley et al., 2012).

- 53 -

Figure 15. Genomic details of Hungarian bat protoparvoviruses. Conserved amino-acid motifs are described as well. Possible recombination event crossing point is also indicated.

- 54 -

86 mouse parvovirus 1 (U12469) mouse parvovirus 4 (FJ440683) 100 hamster parvovirus (U34255)

100 58 mouse parvovirus 5 (FJ441297) 100 mouse parvovirus 2 (DQ196319) 100 LuIII virus (M81888) 61 minute virus of mice (immunos.) (M12032) Rodent minute virus of mice (prototype) (J02275) protoparvovirus 1 100 100 minute virus of mice (Missouri) (DQ196317) 79 minute virus of mice (Cutter) (U34256)

100 rat minute virus 1 (AF332882) 100 H-1 parvovirus (X01457) 100 Kilham rat virus (AF321230) Rodent 99 rat parvovirus 1 (AF036710) protoparvovirus 2 100 porcine parvovirus NADL-2 (L23427) Ungulate porcine parvovirus Kresse (U44978) protoparvovirus 1 95 feline parvovirus (EU659111) mink enteritis virus (D00765) Carnivore 100 protoparvovirus 1 71 canine parvovirus (M19296) 89 racoon parvovirus (JN867610)

100 bufavirus 1a (JX027296) 80 bufavirus 1b (JX027295) Primate 100 bufavirus 3 (KM580348) protoparvovirus 1 100 bufavirus 2 (JX027297) WUHARV parvovirus 1 (JX627576) 100 bat parvovirus (KC154060)

100 bat parvovirus (KC154061) 93 bat bufavirus 1 (KR078343) 100 bat bufavirus 1 (KR078344) chicken parvovirus (GU214704)

0.1

Figure 16. The tree was constructed based on a nucleic acid sequences 3394 bp coding region of the genome. Bat-derived parvoviruses detected previously by others are indicated in grey. Hungarian BtBV strains identified in this study are marked in boldface.

We performed a recombination analysis in order to reveal any recombination events related to our sequences. As a result, a major recombination event was described (Fig. 17), which traces the origins of WUHARV parvovirus from human and bat-related bufaviruses. Further recombinant, bat-related parvoviruses were recently described in Indonesian megabats. The most common recent ancestor of megabat bufavirus 1 might have arisen from multiple genetic recombination events (Sasaki et al., 2016). These studies well reflect the importance of recombination in the evolution of this group of viruses.

- 55 -

Figure 17. Recombination analyses within bufaviruses. A Simplot (A) analysis revealed significant recombination between the existing bat (blue line) and human (red line) bufavirus strains. Phylogenetic analyses performed with sequence data before (B) and after (C) the hypothetic crossing-point verified the possible recombination event.

Viral metagenomic analyses of M. schreibersii bats provide the first sequence data of bat parvoviruses from Europe with phylogenetic relatedness to the recently described human- infecting bufaviruses. The discovery of this novel BtBV along with the detailed molecular and recombination analyses expands our knowledge on the evolutionary history of bufaviruses and widens the list of human-infecting viruses with possible bat origin. The shared evolutionary history between primate and bat bufaviruses seems to be interesting; however, the zoonotic transmission potential and the possible health risk posed by the newly described virus remains to be clarified. Our results revealed the evolutionary origins of a primate protoparvovirus, suggesting a possible recombination event in the past, which highlights the ability of these viruses to cross species barriers.

- 56 -

CRESS DNA viruses (Genomoviridae and Circoviridae) In this project, a total of 21 bat species were tested from Hungary, Georgia, Romania, Serbia and Ukraine. CRESS DNA viruses were identified in the following 8 species: Myotis alcathoe, Myotis daubentonii, Myotis emarginatus, Myotis nattereri, Nyctalus noctula, Pipistrellus nathusii, Pipistrellus pygmaeus, Plecotus auritus. Based on next-generation sequence data and consensus PCR screening, altogether nine complete genomic sequences were amplified with iPCR method and characterized, detected in eight guanos samples. Two full-length genomes represent the Genomoviridae family with 2157 and 2155 nucleotide length. Six members of the family Circoviridae have been described with a full-length genome size ranging from 1638 to 2017 nucleotides. Moreover, Niminivirus has been also detected, representing a Geminivirus-like viral strain with a complete genome size of 2383 nucleotides. For detailed genomic features see Table 3, where conserved amino-acid motifs, nonanucleotide motifs, GC-content and strain names are summarized. Interestingly GRS motif reported for Geminiviridae family (Nash et al., 2011) has been described in Genomoviridae members (GenBank Accessions: KY302866, KY302867) reported here and in the newly described Niminivirus strain (GenBank Accession: KY302868) also. Other studies also described the presence of geminivirus-like Rep sequence GRS motif previously (Sikorski et al., 2013). Two directional pairwise identity matrix was performed for Circoviridae family members of this study (GenBank Accessions: KY302872, KY302871, KY302870, KY302864, KY302865, KY302869), as described in materials and methods section. Based on the latest ICTV regulations, 80% parvise identity demarcation level indicates novel viral species within the family. Based on this analysis (Supplementary Material), five novel viral species within the family Circoviridae have been characterized: (BO4381/Srb/2014; EP38/Hun/2013; Bb1/Hun/2013 UK02/Ukr/2014; UK03/Ukr/2014). Amino acid sequence identity of the highly conserved replicase region is ranging from 44 to 94 % regarding representatives of the family Circoviridae. A Gemini-like virus has also been described with a 99 % amino acid identity of the replicase gene to the previously described Niminivirus. Two novel members of the family Genomoviridae were further described (BS50/Hun/2013; M64g/Hun/2013) with 61 and 62% identity regarding the amino acid sequence of replicase gene to Faecal-associated gemycircularvirus 5 detected in New Zealand, 2011 (Sikorski et al., 2013).

- 57 -

Table 3: Details of basic genomic features and summary of conserved amino acid motifs found in CRESS DNA viruses described in this study.

Genome Nonanucleotide RCR- Walker Strain name size %GC motif RCR-I RCR-II III Walker A Walker B C V16/Hun/2013 2383 41.5 TAATATTAC FLTYPQ PHFHA YVTK GPSKTGKS YLVLDD VCSN FLVTYP BS50/Hun/2013 2157 52 TAATATTAT Q THLHV YACK GESLTGKT YAVIDD LDDN FLVTYP M64g/Hun/2013 2155 52.1 TAATATTAT Q THLHV YACK GESLTGKT YAVIDD LDDN M64/Hun/2013 1752 42.2 TAATACTAC CFTLNN PHLQG YCSK GPPGSGKS IIDDF ITSN EP38/Hun/2013 1666 42.1 TAGTATTAC CFTLNN PHLQG YCKK GESGTGKT ILDDF - GASGLGK Bb1/Hun/2013 1638 29.9 TAATACTGG CFTLNN PHLQG YCKK T ILDDF TSIN UK03/Ukr/2014 1791 45.5 TAGTATTAC VFTWNN PHLQG YCSK GEPGTGKS IIDDF ITSN UK02/Ukr/2014 1752 44.9 TAATACTAT VFTWNN PHLQG YCSK GLPGTGKS IIDDF ITTN GKPGTGK B04381/Srb/2014 2017 43 TAATACTAG CFTLNN PHLQG YCTK T ILDDF ITSN

The genetic diversity of the viral strains published in this study are further indicated in Fig. 17a, b phylogenetic trees. Strain EP38/Hun/2013; Bb1/Hun/2013; M64/Hun/2013; Uk2/Ukr/2014 and Uk3/Ukr/2014 clustered with Cyclovirus and recently proposed Krikovirus genus members. BO4381/Srb/2014 clearly formed a separate paraphyletic clade, which branches as an outgroup of Circovirus (Fig. 17a). BS50/Hun/2013 and M64g/Hun/2013 formed a separate sister clade of Faecal-associated gemycircularvirus 5 group (Fig. 17b). NCA analysis has been previously used for estimating possible host organisms for different virus groups with high substitution rate (Ng et al., 2012). Therefore we performed a NCA in order to estimate the possible host group of the viral strains within Circoviridae family described in this study (Fig. 18). Based on our NCA host prediction, the Circoviridae family member sequences were grouped as invertebrate and mammalian originate. Strain UK3/Ukr/2014 clearly separated with invertebrate host members, while UK2/Ukr/2014 and M64/Hun/2013 grouped with mammalian Circoviridae members. We were unable to classify B04381/Srb/2014, Bb1/Hun/2013 and EP38/Hun/2013 strains, which can be a result of the high divergence of these strains from other members of the family with clarified host organisms (Fig. 18). Results are shown as canonical score plot, wherein the values for the two most significant contributory factors determined for classification are plotted for both the control and test sequences. 91,1% of original grouped cases correctly classified, which strongly supports the results provided for our novel sequences.

- 58 -

As far as we know, this work provides the first NCA-based host classification for European circovirus sequences. Of course, this prediction needs further confirmation with in vitro or in vivo experiments to reveal the true host scale of these viruses. Members of the family Genomoviridae were not subjected to NCA analysis due to the lack of reference sequence data with clarified host organisms. Among our sequences, strain B04381/Srb/2014 showed the lowest amino acid sequence similarity of 44 % to its closest relative, thus represents a distant member of the family Circoviridae. Although showing Circoviridae-specific genomic characteristics (Table 3), distant relatedness to other members of the Circovirus genus is well represented on the phylogenetic tree by constituting a clearly separate paraphyletic branch (Fig 17a). Regarding previously published classification nomination of 78% nucleic acid identity threshold for species divergence within family Genomoviridae (Sikorski et al., 2013), we considered BS50/Hun/2013 and M64g/Hun/2013 strains as a novel viral species of the family. Since they showed 62 and 61 % amino acid identity, respectively to the closest relative of Faecal-associated gemycircularvirus 5 regarding the replicase gene, BS50/Hun/2013 and M64g/Hun/2013 moreover formed a monophyletic group (Fig 17b) with Faecal-associated gemycircularvirus 5 group, these data may support the assumption for a novel viral species. Strain V16/Hun/2013 showed 99 % nucleic acid sequence identity to Niminivirus, which was previously detected in raw sewage samples from Nigeria (Ng et al., 2012). Sewage samples are rich in plant viruses, possibly reflecting the diversity of local plants consumed by local residents and animals (Ng et al., 2012). Likewise raw sewage, bat guano samples are also well representing the invertebrate or even plant-infecting viruses which are circulating locally in the ecosystem (Kemenesi et al., 2016; Wu et al., 2016). Although it is not clear if viral particles present in guano are infectious as described previously in the case of pepper mild mottle virus, which passed through the human gastrointestinal tract and remained infective (Zhang et al., 2006). UK3/Ukr/2014 presented the greatest homology with a Japanese cycloviral sequence isolated from Taiwan squirrels (Callosciurus erythraeus thaiwanensis) intestinal matter (Sato et al., 2015). This may verify the assumption on invertebrate origin, regarding the dominantly and occasionally insectivorous feeding behaviour of bats and squirrels, respectively (Tamura et al., 1989).

- 59 -

Based on the greatest homology of Rep gene (83%), the closest relative of EP38/Hun/2013 strain is a mosquito-related cyclovirus detected previously in the USA (Ng et al., 2011). This may suppose the mosquito origin of our strain, which needs to be verified by further studies conducted on mosquito samples. However, regarding the NCA result and considering the insectivorous feeding patterns of these bat species, these strains may represent invertebrate viruses with yet undiscovered origins. Further surveillance studies of local invertebrate populations may reveal the exact host organisms of these geographically and evolutionary distantly related virus strains. Furthermore, our results may indicate the widespread presence of CRESS DNA viruses in Europe, which are possibly also widely represented in the ecosystem. For assessing the host range, distribution and epizootiology of these viruses, further studies are necessary in association with invertebrate, vertebrate and plant taxa.

- 60 -

- 61 -

65 Columbid circovirus isolate zj1 DQ090945 99 Chimpanzee stool avian-like circovirus Chimp17 GQ404851 75 Raven circovirus isolate 4-1131 NC_008375

100 Beak and feather disease virus isolate BFDV_DL10B KF768552 85 Gull circovirus JQ685854 Duck circovirus Fujian/2010 HG532019 55 100 Goose circovirus strain yk4 DQ192281

Rhinolophus ferrumequinum circovirus 1 JQ814849 Circoviruses

100 Mink circovirus strain MiCV-DL13 KJ020099 86 Bat circovirus isolate XOR JX863737 99 99 Bat circovirus isolate XOR7 NC_021206

100 Porcine circovirus type 2 strain NB0701 EU727546 85 99 Porcine circovirus 1 AY660574 Barbel circovirus isolate BaCV2 JF279961 61 Silurus glanis circovirus isolate H6 JQ011378 B04381/Srb//2014 KY302870

85 Cyclovirus TN25 GQ404857 60 98 Cyclovirus PKgoat21/PAK/2009 NC_014927 100 Cyclovirus VN isolate hcf2 KF031466 74 Human cyclovirus VS5700009 NC_021568 Feline cyclovirus KM017740

97 UK2/Ukr/2014 KY302872 89 83 Dragonfly cyclovirus isolate DfCyV-A1_To-pool2BNZ13-Pf-2010 HQ638066 Cyclovirus NG14 GQ404855 Bat cyclovirus GF-4c HM228874 96 56 M64/Hun/2013 KY302865 76 Cyclovirus Chimp11 GQ404849 Cyclovirus PK5034 GQ404845 Cycloviruses Bb1/Hun/2013 KY302869

100 Circoviridae batCV-SC703 JN857329 97 Bat circovirus ZS/Yunnan-China/2009 JN377580

99 Uncultured circovirus isolate SDWAP I HQ335042 96 EP38/Hun/2013 KY302864 Mosquito circovirus strain B19 KM972725 100 Dragonfly cyclovirus 2 isolate FL1-NZ38-2010 NC_023869

100 Florida woods cockroach-associated cyclovirus GS140 NC 020206 97 UK3/Ukr/2014 KY302871

0.5

- 62 -

Figure 17a. The maximum-likelihood tree was conducted based on complete genome nucleic acid sequences with representative members of the genera Circovirus, Cyclovirus within the family Circoviridae. Sequences presented in our study are indicated with black dots.

Figure 17b. The maximum-likelihood tree was conducted based on complete genome nucleic acid sequences with representative members of the family Genomoviridae. Geminiviridae family was used as an outgroup. Sequences presented in our study are indicated with black dots.

Figure 18. Canonical score plot of discriminant analysis used to classify viral sequences (reference sample number excluding our samples were 183) into groups by using 4 mononucleotide and 16 dinucleotide frequencies. The graph shows the separation of host groups (mammal, bird, and invertebrate) by use of the two most influential factors. Points represent values for individual sequences. Samples of our study are indicated as grey dots. Table summarizes the classification results of the analysis in details.

- 63 -

Supporting table for Figure 18: Classification results and values are described in this table, supporting NCA of this study.

Classification Results Predicted Group Membership

Host bird invertebrate mammal unknown Total Original Count bird 62 0 4 0 66 invertebrate 2 41 0 0 43 mammal 1 5 67 1 74 unknown 0 1 2 3 6 % bird 93,9 0,0 6,1 0,0 100,0 invertebrate 4,7 95,3 0,0 0,0 100,0 mammal 1,4 6,8 90,5 1,4 100,0 unknown 0,0 14,3 42,9 42,9 100,0 a. 91,1% of original grouped cases correctly classified.

- 64 -

Filoviridae Regarding the mortality events of M. schreibersii bats, likely as a result of Lloviu filovirus (Negredo et al., 2011) infection, we established a surveillance system with Hungarian bat conservationists to frequently monitor known colonies of this species. In 2013, a mass-mortality incidence occurred in a M. schreibersii bat colony causing the death of approximately 500 individuals in Northeast Hungary (Bükk mountain). After investigating the scene, only carcass residuals could be collected not permitting any virological investigation. Other known bat colonies in the area were also checked and found unaffected at this year. Three years later in 2016, five individuals of Schreiber's bats were reported with gross pathologic (hemorrhages) changes (Figure 19.) in Northeast Hungary approximately 50 km far from the site of the 2013 incidence. Interestingly, all five individuals were located close to the entrance of the cave, whilst a huge unaffected colony was hibernating in the depth of the cave separated from them. This may suggest a separate arrival of the infected bats to the cave.

Figure 19. Photo of bat carcasses at the sampling site in 11th February 2016 taken by S. Boldogh.

Hemorrhagic symptoms are visually detectable on all specimens. LLOV RNA was detected in the lung sample of the best-conditioned individual (first from the left).

- 65 -

Carcasses were collected on dry ice and transported to the BSL-4 facility at the Szentágothai Research Centre, University of Pécs for further examination. Briefly, after dissection tissue samples were subjected to homogenization and nucleic acid extraction procedures performed with QIAamp Viral RNA Mini Kit (Qiagen, Germany) according to the manufacturers’ recommendations. Nested RT-PCR specific for the RNA-dependent RNA polymerase gene was used to identify filovirus RNA as described previously (He et al., 2015). The RT-PCR amplicon was purified from agarose gel, then bidirectionally sequenced on ABI Prism 310 Genetic Analyzer. Filovirus RNA was detected in one lung tissue sample. Not surprisingly, successful RT- PCR reaction derived from the apparently freshest carcass collected in the cave (Figure 19.). We suppose that bad condition of bat carcasses and/or degradation of viral nucleic acids might be a possible explanation for the negative results in case of the other four lung samples examined. This rapid degradation and difficult diagnosis of LLOV nucleic acid were reported before (Negredo et al., 2011). Based on Basic Local Alignment Tool (BLAST) search (www.ncbi.nlm.nih.gov) the Hungarian virus (Accession number: MF996369) was most closely related to LLOV strain MS-Liver-86/2003 (JF828358) with 98 % nucleic acid homology. Further phylogenetic analysis supported the previous results of the nucleotide homology search that the Hungarian filovirus represents a novel strain of LLOV (LLOV/Mad/2016/Hungary) within the genus Cuevavirus (Figure 20.). Further experiments such as whole genome amplification and in vitro isolation are currently ongoing in our laboratory.

- 66 -

97 AF086833 EBOV Zaire 1976 96 AF499101 EBOV Zaire KJ660346 EBOV Makona 2014 FJ217162 EBOV Cote d'Ivoire 1994 96 FJ217161 EBOV Bundibugyo 2007 Ebolavirus FJ621583 EBOV Reston 2008 74 99 FJ621585 EBOV Reston 2008 AY729654 EBOV Sudan 2000 98 EU338380 EBOV Sudan 2004 MF996369 Lloviu virus Hungary 2016 Cueavavirus 99 JF828358 Lloviu virus Spain 2003 JX458857 MARV 2009

100 FJ750954 MARV Lake Victoria 2009 Marburgvirus 72 FJ750953 MARV Lake Victoria 2007

0.5

Figure 20. Phylogenetic analysis was performed based on a 346 nucleotide partial sequence of RNA- dependent RNA polymerase gene with the Maximum Likelihood method based on the Tamura 3-parameter model (+G+I). For the maximum-likelihood tree, the best-fit nucleotide substitution model was selected based on the Bayesian information criterion as implemented in the MEGA software. The sequence of this study is indicated with bold letters.

After more than a decade of the mass mortality of M. schreibersii bats in Spain (Cueva del Lloviu), LLOV was re-emerged in Europe, when it caused significant deaths in Hungary among the same bat species. This incidence raises the question, whether it was a second introduction of LLOV to Europe or a continuous silent circulation of the virus among European bats. In order to clarify the exact source of LLOV, further investigations involving the bat- connected ecological niches are needed. Similar situation with great ecological losses was observed in the case of White Nose Syndrome in North-America (Frick et al., 2010). To our best knowledge, this discovery represents the first filovirus sequence reported from the territory of Hungary and represents a significant geographic range expansion of LLOV as well. Considering the Spanish and Hungarian mortality events of these bats, this agent may represent a serious risk for conservation efforts of this bat species, moreover, the human pathogenic role should be also considered and intensively examined in the near future.

- 67 -

Concluding Remarks As seen in several cases during the past decades, bats are now considered as a significant source of emerging diseases. The SARS coronavirus pandemic in 2002 or the recent West-African Ebolavirus epidemic are remarkable examples of this fact. These animals are natural hosts and reservoirs numerous viral taxa as a result of co-evolutionary past. Now bats are considered as key players for viral evolution by hosting the gene-pool of several virus groups. As humanity proceeds on the road of globalization, many factors are leading for increased number of spillover events between bats and other animals or humans. In parallel with these events, the scientific interest has significantly grown and a novel, interdisciplinary research area emerged on the basis of One Health concept. The One Health concept is a worldwide strategy for expanding interdisciplinary collaborations and communications in all aspects of health care for humans, animals and the environment. Based on this initiative we established a large-scale collaboration network in Europe with bat conservationists in order to examine the role of European bat species for viral evolution. We investigated major virus families and applied virus discovery efforts as well with next-generation sequencing. As a result we provided information about the enormous diversity of viruses (Astroviruses, Coronaviruses, CRESS DNA viruses), important recombination events (Bufavirus) and discovered several novel viral species for science (Rotavirus, Calicivirus, etc.). More interestingly we identified an emerging Filovirus (Lloviu virus) in Hungary for the first time. To our best knowledge this virus represents a serious risk for bat conservation efforts and population stability of Miniopterus bats, and in addition human health relevance is also possible. Data provided in this thesis may serve as a strong basis for future research efforts. Several different research topics are ongoing on the basis of these results in our laboratory. Miniopterus schreibersii demonstrated outstanding prevalence rates and viral diversity compared to other species in the study. We intend to better understand the host organism by comparative genomics in this case. The emergence of Lloviu virus is of great interest for science since its discovery. The opportunity for studying this agent in our laboratory opened a variety of connecting research, including specific field examination of bats and biosafety laboratory work as well.

- 68 -

References

Adams MJ, Lefkowitz EJ, King AMQ et al. Changes to taxonomy and the International Code of Virus Classification and Nomenclature ratified by the International Committee on Taxonomy of Viruses Archives of Virology 162: 2505. (2017)

Agnarsson I, Zambrana-Torrelio CM, Flores-Saldana NP, et al. A time-calibrated species-level phylogeny of bats (Chiroptera, Mammalia). PLOS Currents Tree of Life. 1st Edition. (2011)

Alsahafi AJ, Cheng AC. The epidemiology of Middle East respiratory syndrome coronavirus in the Kingdom of Saudi Arabia, 2012–2015. International Journal of Infectious Diseases 45: 1-4. (2016)

Altay A, Yahiro T, Bozdayi G, et al. Bufavirus genotype 3 in Turkish children with severe diarrhoea. Clinical Microbiology and Infection 21: 965.e1-4. (2015)

Annan A, Baldwin HJ, Corman VM, et al. Human betacoronavirus 2c EMC/2012-related viruses in bats, Ghana and Europe. Emerging Infectious Diseases 19: 456-459. (2013)

Anthony SJ, Epstein JH, Murray KA, et al. A strategy to estimate unknown viral diversity in mammals. MBio 4:e00598-13. (2013)

Anthony SJ, Gilardi K, Menachery VD, et al. Further Evidence for Bats as the Evolutionary Source of Middle East Respiratory Syndrome Coronavirus. MBio. 4:e00373-17. (2017)

Appleton H, Higgins PG. Viruses and gastroenteritis in infants. Lancet 1: 1297. (1975)

Asano KM, Gregori F, Hora AS, et al. Group A rotavirus in Brazilian bats: description of novel T15 and H15 genotypes. Archives of Virology 161: 3225–3230 (2016)

August TA, Mathews F, Nunn MA. Alphacoronavirus detected in bats in the United Kingdom. Vector Borne and Zoonotic Diseases 12: 530-533. (2012)

Badrane H, Tordo N. Host switching in lyssavirus history from the Chiroptera to the Carnivora orders. Journal of Virology 75: 8096-8104. (2001) Bányai K, Kemenesi G, Budinski I, et al. Candidate new rotavirus species in Schreiber's bats, Serbia. Infection Genetics and Evolution 48: 19-26. (2017)

Biagini P, Bendinelli M, Hino S, et al. Family Circoviridae. In: Virus Taxonomy - Ninth Report of the International Committee on Taxonomy of Viruses, Edited by King AMQ, Adams MJ, Carstens EB and Lefkowitz EJ. Academic Press, London. pp. 343-346. (2011)

Brandão PE, Scheffer K, Villarreal LY, et al. A coronavirus detected in the vampire bat Desmodus rotundus. Brazilian Journal of Infectious Diseases 12: 466-468. (2008)

Brown KE. The expanding range of parvoviruses which infect humans. Reviews in Medical Virology 20: 231-244. (2010)

- 69 -

Buckwalter MR, Nga PT, Gouilh MA, et al. Identification of a novel neuropathogenic Theiler’s murine encephalomyelitis virus. Journal of Virology 85: 6893-6905. (2011)

Burk R, Bollinger L, Johnson JC, et al. Neglected filoviruses. FEMS Microbiology Reviews 40: 494-519. (2016)

Charif D, Lobry JR. SeqinR 1.0-2: a contributed package to the R project for statistical computing devoted to biological sequences retrieval and analysis. Structural approaches to sequence evolution: Molecules, networks, populations, URL: http://cran.R- project.org/package=seqinr. (2007)

Chen HH, Kong WP, Roos RP. The leader peptide of Theiler’s murine encephalomyelitis virus is a zinc-binding protein. Journal of Virology 69: 8076-8078. (1995)

Chen YN, Phuong VN, Chen HC, et al. Detection of the Severe Acute Respiratory Syndrome- Related Coronavirus and Alphacoronavirus in the Bat Population of Taiwan. Zoonoses Public Health 63: 608-615. (2016)

Chieochansin T, Vutithanachot V, Theamboonlers A, et al. Bufavirus in fecal specimens of patients with and without diarrhea in Thailand. Archives of Virology 160: 1781-1784. (2015)

Chu DK, Poon LL, Guan Y, et al. Novel astroviruses in insectivorous bats. Journal of Virology 82: 9107-9114. (2008)

Choi JY. An outbreak of Middle East Respiratory Syndrome coronavirus infection in South Korea, 2015. Yonsei Medical Journal 56: 1174-1176. (2015)

Coltart CEM, Lindsey B, Ghinai I, et al. The Ebola outbreak, 2013–2016: old lessons for new epidemics. Philosophical Transactions of the Royal Society B: Biological Sciences 372: 20160297. (2016)

Cotmore SF, Agbandje-McKenna M, Chiorini JA, et al. The family Parvoviridae. Archives of Virology 159: 1239-1247. (2014)

Corman VM, Rasche A, Diallo TD, et al. Highly diversified coronaviruses in neotropical bats. Journal of General Virology 94: 1984-1994. (2013)

Dayaram A, Opong A, Jaschke A, et al. Molecular characterisation of a novel cassava associated circular ssDNA virus. Virus Research 166: 130-135. (2012)

De Benedictis P, Schultz-Cherry S, Burnham A, et al. Astrovirus infections in humans and animals - Molecular biology, genetic diversity, and interspecies transmissions. Infect. Genet. Evol. 11: 1529-1544. (2011)

De Wit E, van Doremalen N, Falzarano D, et al. SARS and MERS: recent insights into emerging coronaviruses. Nature Reviews Microbiology 14: 523-534. (2016)

Decaro N, Desario C, Parisi A, et al. Buonavoglia. Genetic analysis of canine parvovirus type 2c. Virology 385: 5-10. (2009)

- 70 -

Dietz C, von Helversen O. Illustrated identification key to the bats of Europe. http://biocenosi.dipbsf.uninsubria.it/didattica/bat_key1.pdf (2004)

Drexler JF, Gloza-Rausch F, Glende J, et al. Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences. Journal of Virology 84: 11336-11349. (2010)

Drexler JF, Corman VM, Wegner T, et al. Amplification of emerging viruses in a bat colony. Emerging Infectious Diseases 17: 449-456. (2011)

Drexler JF, Corman VM, Lukashev AN, et al. Hepatovirus Ecology Consortium - Evolutionary origins of hepatitis A virus in small mammals. Proceedings of the National Academy of Sciences USA 112: 15190-15195. (2015)

Dufkova L, Straková P, Širmarová J, et al. Detection of diverse novel bat astrovirus sequences in the Czech Republic. Vector Borne and Zoonotic Diseases 15: 518-521. (2015)

Duke GM, Hoffman MA, Palmenberg AC. Sequence and structural elements that contribute to efficient encephalomyocarditis virus RNA translation. Journal of Virology 66: 1602-1609. (1992)

Duque H, Palmenberg AC. Phenotypic characterization of three phylogenetically conserved stem-loop motifs in the mengovirus 3′ untranslated region. Journal of Virology 75: 3111-3120. (2001)

Esona MD, Mijatovic-Rustempasic S, Conrardy C, et al. Reassortant Group A Rotavirus from Straw-colored Fruit Bat (Eidolon helvum). Emerging Infectious Diseases 16: 1844-1852. (2010)

Estes MK, Kapikian AZ. Rotaviruses. In: Fields virology. Edited by Knipe DM, Howley PM, Griffin DE, et al. Lippincott Williams & Wilkins, Philadelphia. pp. 1917-1974. (2007)

Farkas T, Sestak K, Wei C, et al. Characterization of a rhesus monkey calicivirus representing a new genus of Caliciviridae. Journal of Virology 82: 5408-5416. (2008)

Fischer K, Zeus V, Kwasnitschka L, et al. Insectivorous bats carry host specific astroviruses and coronaviruses across different regions in Germany. Infection Genetics and Evolution 37: 108-116. (2016)

Fischer K, Pinho dos Reis V, Balkema-Buschmann A. Bat Astroviruses: Towards understanding the transmission dynamics of a neglected virus family. Viruses 9: 34. (2017)

Frick WF, Pollock JF, Hicks AC, et al. An emerging disease causes regional population collapse of a common North American bat species. Science 329: 679-682. (2010)

- 71 -

Friendly M, Fox J. Candisc: Visualizing Generalized Canonical Discriminant and Canonical Correlation Analysis. R package version 0.7-0. https://CRAN.R-project.org/package=candisc. (2016)

Garigliany MM, Börstler J, Jöst H, et al. Characterization of a novel circo-like virus in Aedes vexans mosquitoes from Germany: evidence for a new genus within the family Circoviridae. Journal of General Virology 96: 915-920. (2015)

Ge X, Li J, Peng C, et al. Genetic diversity of novel circular ssDNA viruses in bats in China. Journal of General Virology 92: 2646-2653. (2011)

Ge XY, Li JL, Yang XL, et al. Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor. Nature 503: 535-538. (2013)

Giammanco GM, De Grazia S, Tummolo F, et al. Norovirus GII.4/Sydney/2012 in Italy, Winter 2012–2013. Emerging Infectious Diseases 19: 1348-1349. (2013)

Gloza-Rausch F, Ipsen A, Seebens A, et al. Detection and prevalence patterns of group I coronaviruses in bats, northern Germany. Emerging Infectious Diseases 14: 626-631. (2008)

Góes LG, Ruvalcaba SG, Campos AA, et al. Novel bat coronaviruses, Brazil and Mexico. Emerging Infectious Diseases 19: 1711-1713. (2013)

Gombkötő P, Boldogh S. Hosszúszárnyú denevér - Miniopterus schreibersii (Kuhl, 1817). In: Magyarország emlőseinek atlasza [Atlas of mammals of Hungary]. Edited by Bihari Z, Csorba G, Heltai M. Kossuth Press, Budapest. pp: 127-128. (2007)

Gorbalenya AE, Koonin EV, Lai MM. Putative papain-related thiol proteases of positive-strand RNA viruses - Identification of rubi- and aphthovirus proteases and delineation of a novel conserved domain associated with proteases of rubi-, alpha- and coronaviruses. FEBS Letters 288: 201-205. (1991)

Handley SA, Thackray LB, Zhao G, et al. Pathogenic simian immunodeficiency virus infection is associated with expansion of the enteric virome. Cell 151: 253-266. (2012)

Hargitai R, Pankovics P, Kertész AM, et al. Detection and genetic characterization of a novel parvovirus distantly related to human bufavirus in domestic pigs. Archives of Virology 161: 1033-1037. (2017)

Hayman DT. Bats as Viral Reservoirs. Annual Review of Virology 3: 77-99. (2016)

Hayman DT. As the bat flies. Science 354: 1099-1100 (2016)

He B, Zhang Y, Xu L, et al. Identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in China. Journal of Virology 88: 7070-7082. (2014)

- 72 -

He B, Feng Y, Zhang H, et al. Filovirus RNA in Fruit Bats, China. Emerging Infectious Diseases 21: 1675-1677. (2015)

Hon CC, Lam TY, Shi ZL, et al. Evidence of the recombinant origin of a bat severe acute respiratory syndrome (SARS)-like coronavirus and its implications on the direct ancestor of SARS coronavirus. Journal of Virology 82: 1819-1826. (2008)

Hu B, Ge X, Wang LF, et al. Bat origin of human coronaviruses. Virology Journal 12: 221. (2015)

Huang DD, Wang W, Lu QB, et al. Identification of Bufavirus-1 and Bufavirus-3 in feces of patients with acute diarrhea, China. Scientific Reports 5: 13272. (2015)

Huang C, Liu WJ, Xu W, et al. A bat-derived putative cross-family recombinant coronavirus with a reovirus gene. PLoS Pathogens 12:e1005883. (2016)

Hull JJ, Marthaler D, Rossow S, et al. Genomic sequence of the first porcine rotavirus group H strain in the United States. Genome Announcement 4:e01763-15. (2016)

Humphrey W, Dalke A, Schulten K. VMD - Visual Molecular Dynamics. Journal of Molecular Graphics 14: 33-38. (1996)

Hutson AM, Aulagnier S, Benda P, et al. Miniopterus schreibersii. The IUCN Red List of Threatened Species 2008: e.T13561A4160556. http://dx.doi.org/10.2305/IUCN.UK.2008.RLTS.T13561A4160556.en. (2008)

Hutterer R, Ivanova T, Meyer-Cords C, et al. Bat migrations in Europe - A review of banding data and literature. Bundesamt fur Naturschutz, Bonn. pp: 180. (2005)

International Committee on Taxonomy of Viruses - ICTV master species list. http://talk.ictvonline.org/files/ictv_documents/m/msl/5208. (2016)

International Committee on Taxonomy of Viruses - Taxonomy proposal code. http://talk.ictvonline.org/files/proposals/taxonomy_proposals_vertebrate1/m/vert01/4936.asp x. (2014)

Ithete NL, Stoffberg S, Corman VM, et al. Close relative of human Middle East respiratory syndrome coronavirus in bat, South Africa. Emerging Infectious Diseases 19: 1697-1699. (2013)

Jayme SI, Field HE, de Jong C, et al. Molecular evidence of Ebola Reston virus infection in Philippine bats. Virology Journal 12: 107. (2015)

- 73 -

Jiang X, Huang PW, Zhong WM, et al. Design and evaluation of a primer pair that detects both Norwalk- and Sapporo-like caliciviruses by RT-PCR. Journal of Virological Methods 83: 145- 154. (1999)

Jiwaji M, Short JR, Dorrington RA. Expanding the host range of small insect RNA viruses: Providence virus (Carmotetraviridae) infects and replicates in a human tissue culture cell line. Journal of General Virology 97: 2763-2768. (2016)

Johnson KC, Hitchens PL, Smiley ET, et al. Spillover and pandemic properties of zoonotic viruses with high host plasticity. Scientific Reports 5:14830. (2015)

Jones KE, Purvis A, MacLarnon A, et al. A phylogenetic supertree of the bats (Mammalia: Chiroptera). Biological Reviews 77: 223-259. (2002)

Jones KE, Patel NG, Levy MA, et al. Global trends in emerging infectious diseases. Nature 451: 990-993. (2008)

Kapoor A, Victoria J, Simmonds P, et al. A highly prevalent and genetically diversified Picornaviridae genus in South Asian children. Proceedings of the National Academy of Sciences USA 105: 20482-20487. (2008)

Kemenesi G, Dallos B, Görföl T, et al. Novel European lineages of bat astroviruses identified in Hungary. Acta Virologica 58: 95-98. (2014a)

Kemenesi G, Dallos B, Görföl T, et al. Molecular survey of RNA viruses in Hungarian bats: discovering novel astroviruses, coronaviruses, and caliciviruses. Vector Borne and Zoonotic Diseases 14: 846-855. (2014b)

Kemenesi G, Zhang D, Marton S, et al. Genetic characterization of a novel picornavirus detected in Miniopterus schreibersii bats. Journal of General Virology 96: 815-821. (2015a)

Kemenesi G, Dallos B, Görföl T, et al. Genetic diversity and recombination within bufaviruses: detection of a novel strain in Hungarian bats. Infection Genetics and Evolution 33: 288-292. (2015b)

Kemenesi G, Földes F, Kurucz K, et al. Genetic characterization of Providence virus isolated from bat guano in Hungary. Genome Announcement 4:e00403-16. (2016)

Kim HK, Yoon SW, Kim DJ, et al. Detection of Severe Acute Respiratory Syndrome-like, Middle East Respiratory Syndrome-like bat coronaviruses and group H rotavirus in faeces of Korean bats. Transboundary and Emerging Diseases 63: 365-372. (2016)

Knowles NJ, Hovi T, Hyypiä T, et al. Picornaviridae. In: Virus taxonomy - classification and nomenclature of viruses: Ninth Report of the International Committee on Taxonomy of Viruses. Edidet by King AMQ, Adams MJ, Carstens EB, et al. Elsevier, San Diego. pp 855- 880. (2012)

- 74 -

Kohl C, Kurth A. European bats as carriers of viruses with zoonotic potential. Viruses 6: 3110- 3128. (2014)

Krupovic M, Ghabrial SA, Jiang D, et al. Genomoviridae: a new family of widespread single- stranded DNA viruses. Archives of Virology 161: 2633-2643. (2016)

Kunz TH, Hodgkison R, Weise CD. Methods of capturing and handling bats. In: Ecological and Behavioral Methods for the Study of Bats, Edited by Kunz T, Parsons S. Johns Hopkins University Press, Baltimore. pp. 3-35. (2009)

Lau SK, Woo PC, Li KS, et al. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proceedings of the National Academy of Sciences USA 102:14040-5. (2005)

Lau SK, Woo PC, Lai KK, et al. Complete genome analysis of three novel picornaviruses from diverse bat species. Journal of Virology 85: 8819-8828. (2011)

Law KM, Brown TD. The complete nucleotide sequence of the GDVII strain of Theiler’s murine encephalomyelitis virus (TMEV). Nucleic Acids Research 18: 6707-6708. (1990)

Lelli D, Papetti A, Sabelli C, et al. Detection of coronaviruses in bats of various species in Italy. Viruses 5: 2679-2689. (2013)

L'Homme Y, Sansregret R, Plante-Fortier E, et al. Genomic characterization of swine caliciviruses representing a new genus of Caliciviridae. Virus Genes 39: 66-75. (2009)

Li W, Shi Z, Yu M, et al. Bats are natural reservoirs of SARS-like coronaviruses. Science 310: 676-679. (2005)

Li L, Victoria JG, Wang C, et al. Bat guano virome: predominance of dietary viruses from insects and plants plus novel mammalian viruses. Journal of Virology 84: 6955-6965. (2010a)

Li LL, Kapoor A, Slikas B, et al. Multiple diverse circoviruses infect farm animals and are commonly found in human and chimpanzee feces. Journal of Virology 84: 1674-1682. (2010b)

Li L, Pesavento PA, Shan T, et al. Viruses in diarrhoeic dogs include novel kobuviruses and sapoviruses. Journal of General Virology 92: 2534-2541. (2011a)

Li L, Shan T, Soji OB, et al. Possible cross-species transmission of circoviruses and cycloviruses among farm animals. Journal of General Virology 92: 768-772. (2011b)

Liang Z, Kumar AS, Jones MS, et al. Phylogenetic analysis of the species Theilovirus: emerging murine and human pathogens. Journal of Virology 82: 11545-11554. (2008)

Liao Q, Wang X, Wang D, et al. Complete genome sequence of a novel calicivirus from a goose. Archives of Virology 159: 2529-2531. (2014)

- 75 -

Lim ES, Cao S, Holtz LR, et al. Discovery of rosavirus 2, a novel variant of a rodent-associated picornavirus, in children from the Gambia. Virology 454-455: 25-33. (2014)

Lima FE, Cibulski SP, Dos Santos HF, etr al. Genomic characterization of novel circular ssDNA viruses from insectivorous bats in Southern Brazil. PLoS One 10:e0118070. (2015a)

Lima FE, Cibulski SP, Dall Bello AG, et al. A novel chiropteran circovirus genome recovered from a Brazilian insectivorous bat species. Genome Announcement 3:e01393-15. (2015b)

Liu L, Schwarz L, Ullman K, et al. Identification of a novel bufavirus in domestic pigs by a viral metagenomic approach. Journal of General Virology 97: 1592-1596. (2016)

Lole KS, Bollinger RC, Paranjape RS, et al. Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. Journal of Virology 73: 152-160. (1990)

Luis AD, Hayman DTS, O'Shea TJ, et al. A comparison of bats and rodents as reservoirs of zoonotic viruses: are bats special? Proceedings. Biological Sciences 280: 20122753. (2013)

Male MF, Kraberger S, Stainton D, et al. Cycloviruses, gemycircularviruses and other novel replication-associated protein encoding circular viruses in Pacific flying fox (Pteropus tonganus) faeces. Infection Genetics and Evolution 39: 279-292. (2016)

Marchler-Bauer A, Lu S, Anderson JB, et al. CDD: a Conserved Domain Database for the functional annotation of proteins. Nucleic Acids Research 39: D225-D229. (2011)

Marí Saéz A, Weiss S, Nowak K, et al. Investigating the zoonotic origin of the West African Ebola epidemic. EMBO Molecular Medicine 7: 17-23. (2014)

Matthijnssens J, Otto PH, Ciarlet M, et al. VP6-sequence-based cutoff values as a criterion for rotavirus species demarcation. Archives of Virology 157: 1177-1182. (2012)

Medici MC, Tummolo F, Calderaro A, et al. Identification of the novel Kawasaki 2014 GII.17 human norovirus strain in Italy. Euro Surveillence 20:30011. (2015)

Menachery VD, Yount BL Jr, Debbink K, et al. A SARS-like cluster of circulating bat coronaviruses shows potential for human emergence. Nature Medicine 21: 1508-1513. (2015)

Mikalsen AB, Nilsen P, Frøystad-Saugen M, et al. Characterization of a novel calicivirus causing systemic infection in atlantic salmon (Salmosalar L.): proposal for a new genus of caliciviridae. PLoS One 9:e107132. (2014)

Mohd HA, Al-Tawfiq JA, Memish ZA. Middle East Respiratory Syndrome Coronavirus (MERS-CoV) origin and animal reservoir. Virology Journal 13: 87. (2016)

Molnár V, Jánoska M, Harrach B, et al. Detection of a novel bat gamma-herpesvirus in Hungary. Acta Veterinary Hungariae 56: 529-538. (2008)

- 76 -

Muhire BM, Varsani A, Martin DP. Sdt: a virus classification tool based on pairwise sequence alignment and identity calculation. PLoS One 9:e108277. (2014)

Myoung J, Hou W, Kang B, et al. The immunodominant CD8+ T cell epitope region of Theiler’s virus in resistant C57BL/6 mice is critical for anti-viral immune responses, viral persistence, and binding to the host cells. Virology 360: 159-171. (2007)

Nash TE, Dallas MB, Reyes MI, et al. Functional analysis of a novel motif conserved across geminivirus Rep proteins. Journal of Virology 85: 1182-1192. (2011)

Negredo A, Palacios G, Vázquez-Morón S, et al. Discovery of an ebolavirus-like filovirus in europe. PLoS Pathogens 7:e1002304. (2011)

Ng TF, Willner DL, Lim YW, et al. Broad surveys of DNA viral diversity obtained through viral metagenomics of mosquitoes. PLoS One 6:e20579. (2011)

Ng TF, Marine R, Wang C, et al. High variety of known and new RNA and DNA viruses of diverse origins in untreated sewage. Journal of Virology 86: 12161-12175. (2012)

Nyaga MM, Peenze I, Potgieter CA, et al. Complete genome analyses of the first porcine rotavirus group H identified from a South African pig does not provide evidence for recent interspecies transmission events. Infection Genetics and Evolution 38: 1-7. (2016)

Oka T, Wang Q, Katayama K, et al. Comprehensive review of human sapoviruses. Clinical Microbiology Reviews 28: 32-53. (2015)

Ossiboff RJ, Zhou Y, Lightfoot PJ, et al. Conformational changes in the capsid of a calicivirus upon interaction with its functional receptor. Journal of Virology 84: 5550-5564. (2010)

Pevear DC, Calenoff M, Rozhon E, et al. Analysis of the complete nucleotide sequence of the picornavirus Theiler’s murine encephalomyelitis virus indicates that it is closely related to cardioviruses. Journal of Virology 61: 1507-1516. (1987)

Phan TG, Vo NP, Bonkoungou IJ, et al. Acute diarrhea in West African children: diverse enteric viruses and a novel parvovirus genus. Journal of Virology 86: 11024-11030. (2012)

Phan TG, Mori D, Deng X, et al. Small circular single stranded DNA viral genomes in unexplained cases of human encephalitis, diarrhea, and in untreated sewage. Virology 482: 98- 104. (2015)

Pinto P, Wang Q, Chen N, et al. Discovery and genomic characterization of noroviruses from a gastroenteritis outbreak in domestic cats in the US. PLoS One 2:e32739. (2012)

Pringle FM, Johnson KN, Goodman CL, et al. Providence virus: a new member of the Tetraviridae that infects cultured insect cells. Virology 306: 359-370. (2003)

- 77 -

Quan PL, Wagner TA, Briese T, et al. Astrovirus encephalitis in boy with X-linked agammaglobulinemia. Emerging Infectious Diseases 16: 918-925. (2010)

Quetglas J, Gonzalez F, Paz O. Estudian la extraña mortandad de miles de murcielago de cuevas. Quercus 203: 50. (2003) [Spanish]

R Development Core Team R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org (2015)

Rasche A, Souza BF, Drexler JF. Bat hepadnaviruses and the origins of primate hepatitis B viruses. Current Opinion in Virology 16: 86-94. (2016)

Ren W, Li W, Yu M, et al. Full-length genome sequences of two SARS-like coronaviruses in horseshoe bats and genetic variation analysis. Journal of General Virology 87: 3355-3359. (2006)

Reuter G, Pankovics P, Gyöngyi Z, et al. A. Novel dicistrovirus from bat guano. Archives of Virology 159: 3453-3456. (2014)

Reusken CB, Lina PH, Pielaat A, et al. Circulation of group 2 coronaviruses in a bat species common to urban areas in Western Europe. Vector Borne and Zoonotic Diseases 10: 785-791. (2010)

Rihtaric D, Hostnik P, Steyer A, et al. Identification of SARS-like coronaviruses in horseshoe bats (Rhinolophus hipposideros) in Slovenia. Archives of Virology 155: 507-514. (2010)

Rohll JB, Moon DH, Evans DJ, et al. The 3′ untranslated region of picornavirus RNA: features required for efficient genome replication. Journal of Virology 69: 7835-7844. (1995)

Rosario K, Duffy S, Breitbart M. A field guide to eukaryotic circular single-stranded DNA viruses: insights gained from metagenomics. Archives of Virology 157: 1851-1871. (2012)

Rosario K, Breitbart M, Harrach B, et al. Revisiting the taxonomy of the family Circoviridae: establisment of the genus Cyclovirus and removal of the genus Gyrovirus. Archives of Virology 162: 1447-1463. (2017)

Roy A, Kucukural A, Zhang Y. I-TASSER: a unified platform for automated protein structure and function prediction. Nature Protocols 5: 725-738. (2010)

Sasaki M, Orba Y, Anindita PD, et al. Distinct lineages of Bufavirus in wild shrews and nonhuman primates. Emerging Infectious Diseases 21: 1230-1233. (2015)

Sasaki M, Gonzalez G, Wada Y, et al. Divergent bufavirus harboured in megabats represents a new lineage of parvoviruses. Scientific Reports 6: 24257. (2016)

Sato G, Kawashima T, Kiuchi M, et al. Novel cyclovirus detected in the intestinal contents of Taiwan squirrels (Callosciurus erythraeus thaiwanensis).Virus Genes 51: 148-151. (2015)

- 78 -

Scheuer KA, Oka T, Hoet AE, et al. Prevalence of porcine noroviruses, molecular characterization of emerging porcine sapoviruses from finisher swine in the United States, and unified classification scheme for sapoviruses. Journal of Clinical Microbiology 51: 2344-2353. (2013)

Schrödinger L. Schrödinger Suite, Schrödinger, LLC, New York. (2015)

Shackelton LA, Hoelzer K, Parrish CR, et al. Comparative analysis reveals frequent recombination in the parvoviruses. Journal of General Virology 88: 3294-3301. (2007)

Sikorski A, Massaro M, Kraberger S, et al. Novel myco-like DNA viruses discovered in the faecal matter of various animals. Virus Research 177: 209-216. (2013)

Smith AW, Skilling DE, Cherry N, et al. Calicivirus emergence from ocean reservoirs: zoonotic and interspecies movements. Emerging Infectious Diseases 4: 13-20. (1998)

Smits SL, Schapendonk CM, van Beek J, et al. New viruses in idiopathic human diarrhea cases, the Netherlands. Emerging Infectious Diseases 20: 1218-1222. (2014)

Smiley JR, Chang KO, Hayes J, et al. Characterization of an enteropathogenic bovine calicivirus representing a potentially new calicivirus genus. Journal of Virology 76: 10089- 10098. (2002)

Smith NA, Wang LF. Bats and their virome: an important source of emerging viruses capable of infecting humans. Current Opinion in Virology. 3: 84-91. (2013)

Soma T, Nakagomi O, Nakagomi T, et al. Detection of Norovirus and Sapovirus from diarrheic dogs and cats in Japan. Microbiol Immunol 59, 123-128. (2015)

Sorgeloos F, Jha BK, Silverman RH, et al. Evasion of antiviral innate immunity by Theiler’s virus L* protein through direct inhibition of RNase L. PLoS Pathogens 9:e1003474. (2013)

Streicker DG, Turmelle AS, Vonhof MJ, et al. Host phylogeny constrains cross-species emergence and establishment of rabies virus in bats. Science 329: 676-679. (2010)

Tamura N, Hayashi F, Miyashita K. Spacing and kinship in the formosan squirrel living in different habitats. Oecologia 79: 344-352. (1989)

Tamura K, Stecher G, Peterson D, et al. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Molecular Biology and Evolution 30: 2725-2729. (2013)

Tan le V, van Doorn HR, Nghia HD, et al. Identification of a new cyclovirus in cerebrospinal fluid of patients with acute central nervous system infections. MBio 4:e00231-13. (2013)

Tay WT, Soria MF, Walsh T, et al. A brave new world for an old world pest: Helicoverpa armigera (Lepidoptera: Noctuidae) in Brazil. PLoS One 8:e80134. (2013)

- 79 -

Teeling EC, Springer MS, Madsen O, et al. A molecular phylogeny for bats illuminates biogeography and the fossil record. Science 307: 580-584. (2005)

Tortosa P, Dsouli N, Gomard Y, et al. Evolutionary history of Indian Ocean nycteribiid bat flies mirroring the ecology of their hosts. PLoS One 8:e75215. (2013)

Tracy S, Chapman NM, Drescher KM, et al. Evolution of virulence in picornaviruses. Current Topics in Microbiology and Immunology 299: 193-209. (2006)

Tse H, Chan WM, Li KS, et al. Discovery and genomic characterization of a novel bat sapovirus with unusual genomic features and phylogenetic position. PLoS One 4: e34987. (2012)

Varsani A, Krupovic A. Sequence-based taxonomic framework for the classification of uncultured single-stranded DNA viruses of the family Genomoviridae. Virus Evolution 3: vew037 (2017)

Väisänen E, Kuisma I, Phan TG, et al. Bufavirus in feces of patients with gastroenteritis, Finland. Emerging Infectious Diseases 20: 1077-1080. (2014)

Walter CT, Pringle FM, Nakayinga R, et al. Genome organization and translation products of Providence virus: insight into a unique tetravirus. Journal of General Virology 91: 2826-2835. (2010)

Wang LF, Walker PJ, Poon LL. Mass extinctions, biodiversity and mitochondrial function: Are bats ‘special’ as reservoirs for emerging viruses? Current Opinion in Virology 6: 649-657. (2011)

Wang J, Moore NE, Murray ZL, et al. Discovery of novel virus sequences in an isolated and threatened bat species, the New Zealand lesser short-tailed bat (Mystacina tuberculata). Journal of General Virology 96: 2442-2452. (2015)

Wimsatt WA. Biology of bats. Academic, New York, pp. 11-12. (1970)

Wolf S, Reetz J, Otto P. Genetic characterization of a novel calicivirus from a chicken. Archives of Virology 156: 1143-1150. (2011)

Wu Z, Ren X, Yang L, et al. Virome analysis for identification of novel mammalian viruses in bat species from Chinese provinces. Journal of Virology 86: 10999-11012. (2012)

Wu Z, Yang L, Ren X, et al. Deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases. The ISME Journal 10: 609-620. (2016)

Wynne JW, Wang L-F. Bats and Viruses: Friend or Foe? PLoS Pathogens 9:e1003651. (2013)

Xiao J, Li J, Hu G, et al. Isolation and phylogenetic characterization of bat astroviruses in southern China. Archives of Virology 156: 1415-1423. (2011)

- 80 -

Yahiro T, Wangchuk S, Tshering K, et al. Novel human bufavirus genotype 3 in children with severe diarrhea, Bhutan. Emerging Infectious Diseases 20: 1037-1039. (2014)

Yang L, Wu Z, Ren X, et al. Novel SARS-like betacoronaviruses in bats, China, 2011. Emerging Infectious Diseases 19: 989-991. (2013)

Yang L, Wu Z, Ren X, et al. MERS-related betacoronavirus in Vespertilio superans bats, China. Emerging Infectious Diseases 20: 1260-1262. (2014)

Yang XL, Hu B, Wang B, et al. Isolation and characterization of a novel bat coronavirus closely related to the direct progenitor of severe acute respiratory syndrome coronavirus. Journal of Virology 90: 3253-3256. (2015)

Yang S, Liu D, Wang Y, et al. Bufavirus Protoparvovirus in feces of wild rats in China. Virus Genes 52: 130-133. (2016)

Yang XL, Zhang YZ, Jiang RD, et al. Genetically diverse filoviruses in Rousettus and Eonycteris spp. bats, China, 2009 and 2015. Emerging Infectious Diseases 23: 482-486. (2017)

Yu JM, Li XY, Ao YY, et al. Identification of a novel picornavirus in healthy piglets and seroepidemiological evidence of its presence in humans. PLoS One 8:e70137. (2013)

Zhang T, et al. RNA viral community in human feces: prevalence of plant pathogenic viruses. PLoS Biology 4: 108-118. (2006)

Zhang Y. I-TASSER server for protein 3D structure prediction. BMC Bioinformatics 9: 40. (2008)

Zhang GJ, Cowled C, Shi ZL, et al. Comparative analysis of bat genomes provides insight into the evolution of flight and immunity. Science 339: 456-460. (2013)

Zhou C, Zhang S, Gong Q, et al. A novel gemycircularvirus in an unexplained case of child encephalitis. Virology Journal 12: 197. (2015)

Zhu CH, Chu DKW, Liu W, et al. Detection of diverse astroviruses from bats in China. Journal of General Virology 90: 883-887. (2009)

Zuker M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Research 31: 3406-3415. (2003)

- 81 -

Acknowledgements

This work would not have been possible without the intensive support of bat conservation biologists, taxonomists and researchers, I am honoured to have the possibility of working with them. Therefore, I would like to thank: Dr Tamás Görföl (Hungarian Natural History Museum, Budapest); Imre Dombi (Duna Drava National Park); Dr Péter Estók (Eszterházy Károly University of Applied Sciences, Eger); Dr Sándor Boldogh (Aggtelek National Park); Balázs Máté, József Mészáros (Bakony Bat Protection Association); Gömbkötő Péter (Bükk National Park) Dr. Ivana Budinski (University of Belgrade); Szilárd Bücs, Csaba Jére, István Csősz, Farkas Szodoray-Parádi (Romanian Bat Protection Association); Dr. Anton Vlaschenko, Kseniia Kravchenko (Bat Rehabilitation Center of Feldman Ecopark, Ukraine). I would like to thank all my colleagues in the laboratory as well, especially Bianka Dallos who greatly facilitated my scientific work in the lab and also in the field. Special thanks for all other members of the group, without their help none of these result could have been possible: Miklós Oldal, Brigitta Zana, Fanni Földes, Mónika Madai, Dr. Róbert Herczeg, Safia Zeghbib, Dávid Hederics, Dóra Buzás, Lili Geiger, Krisztina Garai and Henrietta Papp. Special thanks for all students (especially Anett Kepner) who made excellent and outstanding work in the lab and helped me in many parts of my scientific work. I also thank further colleagues at the Research Centre for their indispensable scientific and technical assistance (Péter Urbán, Lilla Czuni, Andrea Kovács-Valasek, Csaba Fekete). I must say special thanks for my wife (and colleague as well) for creating the environment for me to make excellent work and live a happy life at the same time. I must further thank my family for helping me through this long way with unlimited support and making the possibility of learning at the university. I have to say too much thanks for my supervisor to fit this page, for giving unlimited opportunities to study, travel, work and get various experiences to become a good scientist. It is a unique opportunity for me to have the chance of working in a BSL-4 laboratory, I have to thank this as well for my supervisor. I am also happy to have the chance of conducting various research topics at the same time – special thanks for everybody on this page and more.

- 82 -

Supplementary Material Summary of positive bat species per region.

Table caption: Summary of positive bat species indicating all tested numbers in contrast to positives. This table not represents prevalence data of these agents, since different methods were used on different sample cohorts and in different time scale. Specific information about each sample cohort is discussed in the main text. No. Of tested animals (No. Country of Investigated Investigated Virus group Bat species Of positives) origin geographic region time period Miniopterus schreibersii 15(12) Myotis bechsteinii 125(5) Myotis daubentonii 81(6) Myotis emarginatus 5(1) Astroviridae Hungary Hungary (46 sites) 2012-2013 Myotis nattereri 37(1) Nyctalus noctula 14(4) Pipistrellus pygmaeus 6(1) Plecotus auritus 29(1) Myotis daubentonii 81(1) Caliciviridae Myotis alcathoe 16(1) Hungary Hungary (46 sites) 2012-2013 Eptesicus serotinus 7(1) Carmotetraviridae Barbastella barbastellus 15(1) Hungary Hungary 2014 Myotis daubentonii 81(1) Myotis myotis 29(1) Myotis nattereri 37(1) Coronaviridae Pipistrellus pygmaeus 6(2) Hungary Hungary (46 sites) 2012-2013 Rhinolophus euryale 3(1) Rhinolophus ferrumequinum 12(1) Rhinolophus hipposideros 3(1) Hungary (Szársomlyó Picornaviridae Miniopterus schreibersii 13(6) Hungary mountain) 2013 Serbia (Beljanica Reoviridae Miniopterus schreibersii 10(5) Serbia mountain) 2014 Hungary (Szársomlyó Parvoviridae Miniopterus schreibersii 13(5) Hungary mountain) 2013 Myotis alcathoe 16(1) Myotis daubentonii 78(1) Myotis emarginatus 5(1) Hungary, CRESS DNA Myotis nattereri 38(1) Georgia, Serbia, Central-Eastern 2013-2014 viruses Nyctalus noctula 30(1) Romania, Europe Ukraine Pipistrellus nathusii 4(1) Pipistrellus pygmaeus 6(1) Plecotus auritus 19(1) Filoviridae Miniopterus schreibersii 5(1) Hungary Northeast-Hungary 2016

- 83 -

Two directional pairwise identity matrix of Circovirus sequences

Figure caption: Two directional pairwise identity matrix of the replication associated protein amino acid sequences of representative circoviruses and cycloviruses. Red arrows represent novel viral species, whilst white arrow indicates novel viral strain presented in this thesis.

- 84 -

Strain-specific oligonucleotides

Table caption: Summary of iPCR oligonucleotides (Genomoviridae and Circoviridae families) and 3’RACE PCR primers used for Mischivirus strain specific genome-segment amplification. In the case of iPCR method, back to back oligonucleotides were designed on the circular genome.

Virus Primer Annealing Purpose Type temperatu of re (°C) PCR Bufavirus BParV-F: 5´ -ATC TGG GGC TTA TTC ATT TGG- 47 screening PCR 3´ BParV-R: 5´ -CGG GTT ATG TCC TTG TTG TAT- 3´ BParV Long-F: 5′-AAA TGG ATG CTT GGT CAT 51 large long- CCA-3′ genome range BParV Long-R: 5′-TTC CTT TGA AGA CCA TGG segment PCR TTC-3′ amplificatio n Circovirus 14UK3_F 5´-AACTGAGGTCTTCTACTACT-3´ 50 complete iPCR 14UK3_R 5´-TTGAAGTCTCTAACGGGAAT-3´ genome amplificatio n Circovirus 14UK2_F 5´-AGAAGCGTTGTACCTATAC-3´ 52 complete iPCR 14UK2_R 5´-GAGATATTCTTTGATTCCACGA-3 genome amplificatio n Circovirus BO4381_F 5´-TGAAGGACAGGGTAATCGAA-3´ 51 complete iPCR BO4381_R 5´-GGTTGCCAAACTCCGTATTC-3´ genome amplificatio n Circovirus Bb1_F: 5'-AGATATGGATCACAAAAACA-3' 50 complete iPCR Bb1_R: 5'-TGGTTCCCAATGTATTCTAT-3' genome amplificatio n Circovirus M64_F: 5'-AAAGTACAAGTGAAAGGAGGAT-3' 50 complete iPCR M64_R: 5'-GTAGGGGTATCGGTCACATA-3' genome amplificatio n Circovirus EP38_F: 5'-TCGTTTTGAGTGTTGTAAAT-3' 50 complete iPCR EP38_R: 5'-AGGTTTCCAAGAGTACATTG-3' genome amplificatio n Genomovi M64g_F 5´-GGGCATTGAGAATAAAAG-3´ 52 complete iPCR rus M64g_R 5´-GCTACTTTCTTGTTACGT-3´ genome amplificatio n Genomovi BS50_F 5'-CCTCAGCAACATCAAAAAATTC-3' 51 complete iPCR rus BS50_R 5´-ATCTCATCACCAAGTTCGGA-3´ genome amplificatio n Picornavir BPIV-RACE_R 5’-GACTCGAGTCGACATCGA-3’ 52 amplificatio 3’ us BPIV-RACE_F 5’-ATGGAACCAGTCGTGGTG-3’ n of the 3’ RAC BPIV-3’RACE end E 5’-GACTCGAGTCGACATCGATTTTTTTTTTTT- 3’

- 85 -

Rotavirus RVJ_VP6_F1 5´- 42; 50 screening nested J GGATTCTCAAATTATCTCCAAC- 3´ PCR RVJ_VP6_R1 5´-GGAAGTTGAATAAAACCTGG- 3´ RVJ_VP6_F2 5´-CGATTACAACATTGCTTC- 3´ RVJ_VP6_R2 5´-GTTCCATTCTAGCTGTATCA- 3´

- 86 -

Nucleotide composition analysis (NCA) raw data

Model used for NCA analysis: bats.mod<-lm(cbind(A,C,G,T,AA,AC,AG,AT,CA,CC,CG,CT,GA,GC,GG,GT,TA,TC,TG,TT)~Host,data=bats) bats.can<-candisc(bats.mod,data=bats) summary(bats.can,scaling=0)

List of the accession numbers of GenBank reference sequences originated from different hosts (bird, invertebrate, mammal) used for control data set in the nucleotide composition analysis.

Bird: AF311295, AF311296, AF311298, AF311299, AF311302, AJ301633, NC_003410, AF536941, AY450442, AY633653, DQ090945, DQ172906, NC_008033, DQ845074, DQ845075, NC_008521, EU056309, EU056310, GQ334371, GQ386944, GU320569, GU936289, HQ738643, HQ738644, NC_014930, HQ180266, HM748938, JQ685854, JQ782207, JX049221, JX221021, JX221024, JX221027, JX221029, JX221040, JX221043, KF031471, KC980909, KF467254, KF561250, KF850537, KF726087, KF723393, KF941315, KF385400, KF385405, KF385412, KF385429, KF385436, KJ704806, KJ866054, KF688551, KF688552, KF688553, NC_025247, KM823541, LC035390, KP229370, KP793918, KM452744, KP943594, KT008265, KT387277, KT454925, KT454926, KT454927

Invertebrate: KC846096, KC846095, JX185421, JX185420, JX185419, NC_023886, HQ638069, HQ638068, HQ638067, HQ638066, HQ638065, HQ638064, HQ638063, HQ638062, HQ638061, HQ638060, HQ638059, HQ638058, HQ638057, HQ638056, HQ638055, HQ638054, HQ638053, HQ638052, HQ638051, HQ638050, HQ638049, NC_023869, JX185423, JX185422, NC_023868, JX185424, KC512917, KC512916, NC_023867, JX185425, NC_023866, JX185427, JX185426, KC512918, KC512919, KC512920, JX569794

Mammal: GQ404844, GQ404845, GQ404846, GQ404847, GQ404848, GQ404849, GQ404850, GQ404854, GQ404855, GQ404857, GQ404858, HQ738634, HQ738635, HQ738636, NC_014927, NC_014928, NC_021568, KF031465, KF031466, KF031467, KF031468, KF031469, KF031470, KF726987, NC_023874, NC_021707, KM017740, NC_024700, KJ831064, KM392286, KM392288, KM392289, KP151567, KR902499, Y09921, U49186, AY219836, AY754015, DQ648032, EF493843, FJ159691, FJ159692, FJ159693, FJ475129, GQ449671, GU371908, GU722334, GQ404852, GQ404853, GU799575, JF690916, JF690922, JF690923, HQ231329, JN133302, JX512856, KC447455, KC835194, KC894933, KF695388, KC859451, KF732649, KF732857, KJ408799, KJ808815, KF887949, KT734826, KT734822, KT734817, KT946839, NC_024694, KJ206566, NC_023885, KJ020099

- 87 -

List of Publications

Publications related to thesis topic Kemenesi G, Kurucz K, Zana B, Földes F, Urbán P, Vlaschenko A, Kravchenko K, Budinski I, Szodoray-Parádi F, Bücs S, Jére C, Csősz I, Szodoray-Parádi A, Estók P, Görföl T, Boldogh S, Jakab F. Diverse replication- associated protein encoding circular DNA viruses in guano samples of Central-Eastern European bats. Arch Virol. 2017 Dec 15. doi: 10.1007/s00705-017-3678-5.

Bányai K*, Kemenesi G*, Budinski I, Földes F, Zana B, Marton S, Kugler R, Oldal M, Kurucz K, Jakab F: Candidate new rotavirus species in Schreiber’s bats, Serbia. Infection Genetics and Evolution 48: 19-26. (2017) *THESE AUTHORS CONTRIBUTED EQUALLY TO THE WORK

Kemenesi G, Földes F, Zana B, Kurucz K, Estók P, Boldogh S, Görföl T, Bányai K, Oldal M, Jakab F: Genetic Characterization of Providence Virus Isolated from Bat Guano in Hungary. Genome Announcements 4:e00403- 16. (2016)

Kemenesi G, Gellért Á, DallosB, Görföl T, Boldogh S, Estók P, Marton Sz, Oldal M, Martella V, Bányai K, Jakab F. Sequencing and molecular modeling identifies candidate members of Caliciviridae family in bats. Infection Genetics and Evolution 41: 227-232. (2016)

Kemenesi G, Dallos B, Görföl T, Estók P, Boldogh S, Kurucz K, Oldal M, Marton Sz, Bányai K, Jakab F. Genetic diversity and recombination within bufaviruses: Detection of a novel strain in Hungarian bats. Infection Genetics and Evolution 33: 288-292. (2015)

Kemenesi G, Zhang D, Marton S, Dallos B, Görföl T, Estók P, Boldogh S, Kurucz K, Oldal M, Kutas A, Bányai K, Jakab F: Genetic characterization of a novel picornavirus detected in Miniopterus schreibersii bats. Journal of General Virology 96: 815-821. (2015)

Kemenesi G, Dallos B, Görföl T, Boldogh S, Estók P, Kurucz K, Kutas A, Földes F, Oldal M, Németh V, Martella V, Bányai K, Jakab F. Molecular Survey of RNA Viruses in Hungarian Bats: Discovering Novel Astroviruses, Coronaviruses, and Caliciviruses. Vector borne and zoonotic diseases 14:846-855. (2014)

Kemenesi G, Dallos B, Görföl T, Boldogh S, Estók P, Kurucz K, Oldal M, Németh V, Madai M, Bányai K, Jakab F. Novel European lineages of bat astroviruses identified in Hungar. Acta virologica 58: 95-98. (2014)

Conference proceedings related to thesis topic Kemenesi G, Zana B, Kurucz K, Vlaschenko A, Kravchenko K, Budinski I, Szodoray F, Görföl T, Bányai K, Jakab F: High diversity of replication-associated protein encoding circular viruses in guano samples of European bats. International Meeting on Emerging Diseases and Surveillance (IMED 2016), Vienna, Austria November 4- 7, 2016 (POSTER)

Kemenesi G., Földes F, Urbán P, Zana B, Kurucz K, Vlaschenko A, Kravchenko K, Budinski I, Szodoray-Parádi F, Szodoray-Parádi A, Bücs Sz, Jére Cs, Csősz I, Estók P, Boldogh S, Görföl T, Bányai K, Jakab F. Cirkuláris egyszálú DNS vírusok genetikai variabilitásának vizsgálata európai denevér mintákban. A Magyar Mikrobiológiai Társaság 2016. évi Naggyűlése, Keszthely, október 19-21, 2016. (SEMIPLENARY PRESENTATION)

Bányai K*, Kemenesi G*, Budinski I, Földes F, Zana B, Marton Sz, Varga-Kugler R, Oldal M, Kurucz K, Jakab F. Új rotavirus leírása szerbiai Hosszúszárnyú denevérekben. A Magyar Mikrobiológiai Társaság 2016. évi Naggyűlése, Keszthely, október 19-21, 2016. (ORAL PRESENTATION) *THESE AUTHORS CONTRIBUTED EQUALLY TO THE WORK

Kemenesi G, Dallos B, Görföl T, Boldogh S, Estók P, Kutas A, Oldal M, Martella V, Bányai K, Jakab F. Molecular survey of bat-transmitted RNA viruses: Discovering novel astroviruses, coronaviruses and caliciviruses. International Meeting on Emerging Diseases and Surveillance (IMED 2014), October 31- November 3., 2014, Vienna, Austria (POSTER)

- 88 -

Publications outside to thesis topic Fehér E, Kemenesi G, Oldal M, Kurucz K, Kugler R, Farkas SL, Marton S, Horváth G, Bányai K, Jakab F: Isolation and complete genome characterization of novel reassortant orthoreovirus from common vole (Microtus arvalis). Virus Genes 53: 307-311. (2017)

Kemenesi G, Kurucz K, Zana B, Tu VT, Görföl T, Estók P, Földes F, Sztancsik K, Urbán P, Fehér E, Jakab F: Highly divergent cyclo-like virus in a great roundleaf bat (Hipposideros armiger), Vietnam. Archives of Virology 162: 2403-2407. (2017)

Kurucz K, Kemenesi G, Zana B, Zeghbib S, Oldal M, Jakab F: Ecological preferences of the putative West Nile virus vector Uranotaenia unguiculata mosquito with description of an original larval habitat. NORTH-WESTERN JOURNAL OF ZOOLOGY e161103 (2017)

Mihalov-Kovács E, Martella V, Lanave G, Bodnar L, Fehér E, Marton S, Kemenesi G, Jakab F, Bányai K. Genome analysis of canine astroviruses reveals genetic heterogeneity and suggests possible inter-species transmission. Virus Research 232: 162-170. (2017)

Zana B, Kemenesi G, Antal L, Földes F, Oldal M, Bányai K, Jakab F: Molecular traces of a putative novel insect flavivirus from Anopheles hyrcanus mosquito species in Hungary. Acta Virologica 61: 127-129. (2017)

Kurucz K, Kiss V, Zana B, Schmieder V, Kepner A, Jakab F, Kemenesi G: Emergence of Aedes koreicus (Diptera: Culicidae) in an urban area, Hungary, 2016 Parasitology Research 115: 4687-4689. (2016)

Zana B, Kemenesi G, Herczeg R, Dallos B, Oldal M, Marton Sz, Krtinic B, Gellért Á, Bányai K, Jakab F: Genomic characterization of West Nile virus strains derived from mosquito samples obtained during 2013 Serbian outbreak Journal of Vector Borne Diseases 53: 379-383. (2016)

Francuski L, Milankov V, Ludoški J, Krtinić B, Lundström JO, Kemenesi G, Jakab F. Genetic and phenotypic variation in central and northern European populations of Aedes (Aedimorphus) vexans (Meigen, 1830) (Diptera, Culicidae). Journal of Vector Ecology 41: 160-171. (2016)

Kurucz K, Kepner A, Krtinic B, Zana B, Földes F, Bányai K, Oldal M, Jakab F, Kemenesi G. First molecular identification of Dirofilaria spp. (Onchocercidae) in mosquitoes from Serbia. Parasitology Research 115: 3257- 3260. (2016)

László Antal, Brigitta László, Petr Kotlík, Attila Mozsár, István Czeglédi, Miklós Oldal, Kemenesi G, Ferenc Jakab, Sándor Alex Nagy: Phylogenetic evidence for a new species of Barbus in the Danube River basin. Molecular Phylogenetics and Evolution 96: 187-194. (2016)

Damásdi M, Jakab F, Kovács K, Oldal M, Kemenesi G, Szabó E, Vályi-Nagy I, Pytel Á, Farkas L, Szántó Á: Prevalence and type diversity of human papillomaviruses in penile cancers in Hungary. Pathology and Oncology Research 22: 643-646. (2016)

Görföl T, Kemenesi G, Jakab F: High diversity of bat-related viruses in Hungary. Magyar Allatorvosok Lapja 137: 679-686. (2015)

Kemenesi G, Kurucz K, Kepner A, Dallos B, Oldal M, Herczeg R, Vajdovics P, Bányai K, Jakab F. Circulation of Dirofilaria repens, Setaria tundra, and Onchocercidae species in Hungary during the period 2011–2013. Veterinary Parasitology 214: 108-113 (2015)

Oldal M, Sironen T, Henttonen H, Vapalahti O, Madai M, Horváth G, Dallos B, Kutas A, Földes F, Kemenesi G, Németh V, Bányai K, Jakab F. Serologic Survey of Orthopoxvirus Infection Among Rodents in Hungary. Vector borne and zoonotic diseases 15: 317-322. (2015)

Kemenesi G, Dallos B, Oldal M, Kutas A, Földes F, Németh V, Reiter P, Bakonyi T, Bányai K, Jakab F. Putative novel lineage of West Nile virus in Uranotaenia unguiculata mosquito, Hungary. VirusDisease 25: 500-503. (2014)

- 89 -

Oldal M, Németh V, Madai M, Pintér R, Kemenesi G, Dallos B, Kutas A, Sebők J, Horváth G, Bányai K, Jakab F. Serosurvey of pathogenic hantaviruses among forestry workers in Hungary. International Journal of Occupational Medicine and Environmental Health 27: 766-773 (2014)

Szentpáli-Gavallér K, Antal L, Tóth M, Kemenesi G, Soltész Z, Dán Á, Erdélyi K, Bányai K, Dán B, Jakab F, Bakonyi T. Monitoring of West Nile Virus in Mosquitoes Between 2011–2012 in Hungary. Vector Borne and Zoonotic Diseases 14: 648-655. (2014)

Pintér R, Madai M, Horváth G, Németh V, Oldal M, Kemenesi G, Dallos B, Bányai K, Jakab F. Molecular Detection and Phylogenetic Analysis of Tick-Borne Encephalitis Virus in Rodents Captured in the Transdanubian Region of Hungary. Vector borne and zoonotic diseases 14: 621-624. (2014)

Kemenesi G, Krtinić B, Milankov V, Kutas A, Dallos B, Oldal M, Somogyi N, Nemeth V, Banyai K, Jakab F. West Nile virus surveillance in mosquitoes, April to October 2013, Vojvodina province, Serbia: Implications for the 2014 season. Eurosurveillance 24;19(16):20779 (2014)

Oldal M, Németh V, Madai M, Kemenesi G, Dallos B, Péterfi Z, Sebők J, Wittmann I, Bányai K, Jakab F. Identification of hantavirus infection by Western blot assay and TaqMan PCR in patients hospitalized with acute kidney injury. Diagnostic microbiology and infectious disease 79(2) (2014)

Németh V, Oldal M, Madai M, Horváth G, Kemenesi G, Dallos B, Bányai K, Jakab F. Molecular characterization of Dobrava and Kurkino genotypes of Dobrava-Belgrade hantavirus detected in Hungary and Northern Croatia. Virus Genes 47: 546-549 (2013)

Pintér R, Madai M, Vadkerti E, Németh V, Oldal M, Kemenesi G, Dallos B, Gyuranecz M, Kiss G, Bányai K, Jakab F. Identification of tick-borne encephalitis virus in ticks collected in southeastern Hungary. Ticks and Tick- borne Diseases 4: 427-431 (2013)

Conference proceedings outside thesis topic Kemenesi G, Kurucz K, Zana B, Tu Vuong Tan, Görföl T, Estók P, Földes F, Sztancsik K, Jakab F. Highly divergent Cyclo-like virus in a Great roundleaf bat (Hipposideros armiger), Vietnam. 8th International Conference on Emerging Zoonoses, Manhattan KS, USA, Május 7-10 2017 (poster)

Kemenesi G, Kepner A, Kurucz K, Jakab F: Dirofilariosis in Hungary, experiences from mosquito surveillance studies in Hungary and Serbia. 1ST VETERINARY ONCOLOGY AND CLINICAL PATHOLOGY MEETING – VISEGRÁD; 06/2016 (oral presentation)

- 90 -

Statement

Nyilatkozat a dolgozat eredetiségéről

Alulírott Kemenesi Gábor (szül: Kemenesi Gábor, an: László Mária Margit, szül. hely, idő: Budapest 1987.09.19.) “Evolutionary relationships of bat-related viruses of European bats” című doktori értekezésemet a mai napon benyújtom a Biológiai és Sportbiológiai Doktori

Iskola részére. Témavezető: Dr. Jakab Ferenc, egyetemi docens.

Egyúttal nyilatkozom, hogy:

 korábban más doktori iskolában (sem hazai, sem külföldi egyetemen) nem nyújtottam be,  fokozatszerzési eljárásra jelentkezésemet két éven belül nem utasították el,  az elmúlt két esztendőben nem volt sikertelen doktori eljárásom,  öt éven belül doktori fokozatom visszavonására nem került sor,  értekezésem önálló munka, más szellemi alkotását sajátomként nem mutattam be, az irodalmi hivatkozások egyértelműek és teljesek, az értekezés elkészítésénél hamis vagy hamisított adatokat nem használtam.

Dátum:......

Doktorjelölt aláírása

- 91 -