UNIVERSITY OF PÉCS

Doctoral School of Biology and Sportbiology

Evolutionary relationships of bat-related viruses of European

bats

PhD Thesis

GÁBOR KEMENESI

PÉCS, 2018

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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

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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 -

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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 -

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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

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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).

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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).

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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).

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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).

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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.

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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.

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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).

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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).

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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).

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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).

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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).

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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).

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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.

- 19 -

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.

- 20 -

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).

- 21 -

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.

- 22 -

A

B

- 23 -

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).

- 24 -

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

amplicon

size (nt)

Type of PCR References

AstV F1/1: 5´ -GARTTYGATTGGRCKCGKTAYGA- 3´ 422 Semi-nested

RT-PCR

Chu et al.,

2008

F1/2: 5´ -GARTTYGATTGGRCKCGKTAYGA- 3´

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-

PCR

de Souza-

Luna et al.,

2007 PC2As1: 5´ -TCATCACTCAGAATCATCA- 3´

PCS: 5´ -

CTTATGGGTTGGGATTATCCTAAGTGTGA- 3´

PCNAs: 5´ -

CACACAACACCTTCATCAGATAGAATCATCA- 3´

FiV FV-F1: 5’ – GCMTTYCCIAGYAAYATGATGG- 3’ 405 nested RT-

PCR

Hu et al.,

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.

- 25 -

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.

- 26 -

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).

- 27 -

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.

- 28 -

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).

- 29 -

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.

- 30 -

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).

- 31 -

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).

- 32 -

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.

- 33 -

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).

- 34 -

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.

- 35 -

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).

- 36 -

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.

- 37 -

- 38 -

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.

- 39 -

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.

- 40 -

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.

- 41 -

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).

- 42 -

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).

- 43 -

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).

- 44 -

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).

- 45 -

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.

- 46 -

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.

- 47 -

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).

- 48 -

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).

- 49 -

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.

Proteins RVA RVB RVC RVD RVF RVG RVH RVI

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

- 50 -

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).

- 51 -

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.

- 52 -

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.

- 53 -

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).

- 54 -

Figure 15. Genomic details of Hungarian bat protoparvoviruses. Conserved amino-acid motifs are

described as well. Possible recombination event crossing point is also indicated.

- 55 -

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.

mouse parvovirus 1 (U12469)

mouse parvovirus 4 (FJ440683)

hamster parvovirus (U34255)

mouse parvovirus 5 (FJ441297)

mouse parvovirus 2 (DQ196319)

LuIII virus (M81888)

minute virus of mice (immunos.) (M12032)

minute virus of mice (prototype) (J02275)

minute virus of mice (Missouri) (DQ196317)

minute virus of mice (Cutter) (U34256)

rat minute virus 1 (AF332882)

H-1 parvovirus (X01457)

Kilham rat virus (AF321230)

Rodent

protoparvovirus 1

Rodent

protoparvovirus 2 rat parvovirus 1 (AF036710)

porcine parvovirus NADL-2 (L23427)

porcine parvovirus Kresse (U44978) Ungulate

protoparvovirus 1 feline parvovirus (EU659111)

mink enteritis virus (D00765)

canine parvovirus (M19296)

racoon parvovirus (JN867610)

Carnivore

protoparvovirus 1

bufavirus 1a (JX027296)

bufavirus 1b (JX027295)

bufavirus 3 (KM580348)

bufavirus 2 (JX027297)

WUHARV parvovirus 1 (JX627576)

bat parvovirus (KC154060)

bat parvovirus (KC154061)

bat bufavirus 1 (KR078343)

bat bufavirus 1 (KR078344)

Primate

protoparvovirus 1

chicken parvovirus (GU214704)

100

100

80

100

93

100

100

100

100

89

100

71

95

99

100

100

100

61

79

100 100

100

100

100

86

58

100

0.1

- 56 -

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.

- 57 -

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).

- 58 -

Table 3: Details of basic genomic features and summary of conserved amino acid motifs found in CRESS

DNA viruses described in this study.

Strain name

Genome

size %GC

Nonanucleotide

motif RCR-I RCR-II

RCR-

III Walker A Walker B

Walker

C

V16/Hun/2013 2383 41.5 TAATATTAC FLTYPQ PHFHA YVTK GPSKTGKS YLVLDD VCSN

BS50/Hun/2013 2157 52 TAATATTAT

FLVTYP

Q THLHV YACK GESLTGKT YAVIDD LDDN

M64g/Hun/2013 2155 52.1 TAATATTAT FLVTYPQ 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 -

Bb1/Hun/2013 1638 29.9 TAATACTGG CFTLNN PHLQG YCKK

GASGLGK

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

B04381/Srb/2014 2017 43 TAATACTAG CFTLNN PHLQG YCTK

GKPGTGK

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.

- 59 -

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).

- 60 -

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.

- 61 -

- 62 -

Columbid circovirus isolate zj1 DQ090945

Chimpanzee stool avian-like circovirus Chimp17 GQ404851

Raven circovirus isolate 4-1131 NC_008375

Beak and feather disease virus isolate BFDV_DL10B KF768552

Gull circovirus JQ685854

Duck circovirus Fujian/2010 HG532019

Goose circovirus strain yk4 DQ192281

Rhinolophus ferrumequinum circovirus 1 JQ814849

Mink circovirus strain MiCV-DL13 KJ020099

Bat circovirus isolate XOR JX863737

Bat circovirus isolate XOR7 NC_021206

Porcine circovirus type 2 strain NB0701 EU727546

Porcine circovirus 1 AY660574

Barbel circovirus isolate BaCV2 JF279961

Silurus glanis circovirus isolate H6 JQ011378

B04381/Srb//2014 KY302870

Cyclovirus TN25 GQ404857

Cyclovirus PKgoat21/PAK/2009 NC_014927

Cyclovirus VN isolate hcf2 KF031466

Human cyclovirus VS5700009 NC_021568

Feline cyclovirus KM017740

UK2/Ukr/2014 KY302872

Dragonfly cyclovirus isolate DfCyV-A1_To-pool2BNZ13-Pf-2010 HQ638066

Cyclovirus NG14 GQ404855

Bat cyclovirus GF-4c HM228874

M64/Hun/2013 KY302865

Cyclovirus Chimp11 GQ404849

Cyclovirus PK5034 GQ404845

Bb1/Hun/2013 KY302869

Circoviridae batCV-SC703 JN857329

Bat circovirus ZS/Yunnan-China/2009 JN377580

Uncultured circovirus isolate SDWAP I HQ335042

EP38/Hun/2013 KY302864

Mosquito circovirus strain B19 KM972725

Dragonfly cyclovirus 2 isolate FL1-NZ38-2010 NC_023869

Florida woods cockroach-associated cyclovirus GS140 NC 020206

UK3/Ukr/2014 KY302871

99 96

97 100

99

97

100

100

85

100

98

100

100

65 99

100

75

100

97

56

76

83

96

99

74

89

60

85

86

85

61

99

55

0.5

Circoviruses

Cycloviruses

- 63 -

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.

- 64 -

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

of this study.

Classification Results

Host

Predicted Group Membership

Total bird invertebrate mammal unknown

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.

- 65 -

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).

- 66 -

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.

- 67 -

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.

FJ750954 MARV Lake Victoria 2009

FJ750953 MARV Lake Victoria 2007

AF086833 EBOV Zaire 1976

AF499101 EBOV Zaire

KJ660346 EBOV Makona 2014

FJ217162 EBOV Cote d'Ivoire 1994

FJ217161 EBOV Bundibugyo 2007

FJ621583 EBOV Reston 2008

FJ621585 EBOV Reston 2008

AY729654 EBOV Sudan 2000

EU338380 EBOV Sudan 2004

MF996369 Lloviu virus Hungary 2016

JF828358 Lloviu virus Spain 2003

JX458857 MARV 2009

99

72 100

98

99

97 96

96

74

0.5

Cueavavirus

Ebolavirus

Marburgvirus

- 68 -

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.

- 69 -

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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.

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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.

Virus group Bat species

No. Of tested

animals (No.

Of positives)

Country of

origin

Investigated

geographic region

Investigated

time period

Astroviridae

Miniopterus schreibersii 15(12)

Hungary Hungary (46 sites) 2012-2013

Myotis bechsteinii 125(5)

Myotis daubentonii 81(6)

Myotis emarginatus 5(1)

Myotis nattereri 37(1)

Nyctalus noctula 14(4)

Pipistrellus pygmaeus 6(1)

Plecotus auritus 29(1)

Caliciviridae

Myotis daubentonii 81(1)

Hungary Hungary (46 sites) 2012-2013 Myotis alcathoe 16(1)

Eptesicus serotinus 7(1)

Carmotetraviridae Barbastella barbastellus 15(1) Hungary Hungary 2014

Coronaviridae

Myotis daubentonii 81(1)

Hungary Hungary (46 sites) 2012-2013

Myotis myotis 29(1)

Myotis nattereri 37(1)

Pipistrellus pygmaeus 6(2)

Rhinolophus euryale 3(1)

Rhinolophus

ferrumequinum 12(1)

Rhinolophus hipposideros 3(1)

Picornaviridae Miniopterus schreibersii 13(6) Hungary

Hungary

(Szársomlyó

mountain) 2013

Reoviridae Miniopterus schreibersii 10(5) Serbia

Serbia (Beljanica

mountain) 2014

Parvoviridae Miniopterus schreibersii 13(5) Hungary

Hungary

(Szársomlyó

mountain) 2013

CRESS DNA

viruses

Myotis alcathoe 16(1)

Hungary,

Georgia, Serbia,

Romania,

Ukraine

Central-Eastern

Europe 2013-2014

Myotis daubentonii 78(1)

Myotis emarginatus 5(1)

Myotis nattereri 38(1)

Nyctalus noctula 30(1)

Pipistrellus nathusii 4(1)

Pipistrellus pygmaeus 6(1)

Plecotus auritus 19(1)

Filoviridae Miniopterus schreibersii 5(1) Hungary Northeast-Hungary 2016

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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.

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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

temperatu

re (°C)

Purpose Type

of

PCR Bufavirus BParV-F: 5´ -ATC TGG GGC TTA TTC ATT TGG-

3´

BParV-R: 5´ -CGG GTT ATG TCC TTG TTG TAT-

3´

47 screening PCR

BParV Long-F: 5′-AAA TGG ATG CTT GGT CAT

CCA-3′

BParV Long-R: 5′-TTC CTT TGA AGA CCA TGG

TTC-3′

51 large

genome

segment

amplificatio

n

long-

range

PCR

Circovirus 14UK3_F 5´-AACTGAGGTCTTCTACTACT-3´

14UK3_R 5´-TTGAAGTCTCTAACGGGAAT-3´

50 complete

genome

amplificatio

n

iPCR

Circovirus 14UK2_F 5´-AGAAGCGTTGTACCTATAC-3´

14UK2_R 5´-GAGATATTCTTTGATTCCACGA-3

52 complete

genome

amplificatio

n

iPCR

Circovirus BO4381_F 5´-TGAAGGACAGGGTAATCGAA-3´

BO4381_R 5´-GGTTGCCAAACTCCGTATTC-3´

51 complete

genome

amplificatio

n

iPCR

Circovirus Bb1_F: 5'-AGATATGGATCACAAAAACA-3'

Bb1_R: 5'-TGGTTCCCAATGTATTCTAT-3'

50 complete

genome

amplificatio

n

iPCR

Circovirus M64_F: 5'-AAAGTACAAGTGAAAGGAGGAT-3'

M64_R: 5'-GTAGGGGTATCGGTCACATA-3'

50 complete

genome

amplificatio

n

iPCR

Circovirus EP38_F: 5'-TCGTTTTGAGTGTTGTAAAT-3'

EP38_R: 5'-AGGTTTCCAAGAGTACATTG-3'

50 complete

genome

amplificatio

n

iPCR

Genomovi

rus

M64g_F 5´-GGGCATTGAGAATAAAAG-3´

M64g_R 5´-GCTACTTTCTTGTTACGT-3´ 52 complete

genome

amplificatio

n

iPCR

Genomovi

rus

BS50_F 5'-CCTCAGCAACATCAAAAAATTC-3'

BS50_R 5´-ATCTCATCACCAAGTTCGGA-3´ 51 complete

genome

amplificatio

n

iPCR

Picornavir

us

BPIV-RACE_R 5’-GACTCGAGTCGACATCGA-3’

BPIV-RACE_F 5’-ATGGAACCAGTCGTGGTG-3’

BPIV-3’RACE

5’-GACTCGAGTCGACATCGATTTTTTTTTTTT-

3’

52 amplificatio

n of the 3’

end

3’

RAC

E

- 86 -

Rotavirus

J

RVJ_VP6_F1 5´-

GGATTCTCAAATTATCTCCAAC- 3´

RVJ_VP6_R1 5´-GGAAGTTGAATAAAACCTGG-

3´

RVJ_VP6_F2 5´-CGATTACAACATTGCTTC- 3´

RVJ_VP6_R2 5´-GTTCCATTCTAGCTGTATCA-

3´

42; 50 screening nested

PCR

- 87 -

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

- 88 -

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

egyszálu 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 Hosszuszárnyu 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)

- 89 -

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)

- 90 -

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)

- 91 -

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 benyujtom a Biológiai és Sportbiológiai Doktori

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

Egyuttal nyilatkozom, hogy:

korábban más doktori iskolában (sem hazai, sem külföldi egyetemen) nem nyujtottam

be,

fokozatszerzési eljárásra jelentkezésemet két éven belül nem utasították el,

az elmult 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:.....................................

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