Retrospective Detection and Complete Genomic Sequencing of Canine morbillivirus in Eurasian Otter (Lutra lutra) Using Nanopore Technology

The Eurasian otter (Lutra lutra) is a piscivorous apex predator in aquatic habitats, and a flagship species of conservation biology throughout Europe. Despite the wide distribution and ecological relevance of the species, there is a considerable lack of knowledge regarding its virological and veterinary health context, especially in Central Europe. Canine morbillivirus (Canine distemper virus (CDV)) is a highly contagious viral agent of the family Paramyxoviridae with high epizootic potential and veterinary health impact. CDV is present worldwide among a wide range of animals; wild carnivores are at particular risk. As part of a retrospective study, lung-tissue samples (n = 339) from Eurasian otters were collected between 2000 and 2021 throughout Hungary. The samples were screened for CDV using a real-time RT-PCR method. Two specimens proved positive for CDV RNA. In one sample, the complete viral genome was sequenced using a novel, pan-genotype CDV-specific amplicon-based sequencing method with Oxford Nanopore sequencing technology. Both viral sequences were grouped to a European lineage based on the hemagglutinin-gene phylogenetic classification. In this article, we present the feasibility of road-killed animal samples for understanding the long-term dynamics of CDV among wildlife and provide novel virological sequence data to better understand CDV circulation and evolution

Predation of common wall lizards: experiences from a study using scentless plasticine lizards

The potential influence of predators on lacertid lizards has been studied by using models made of plasticine which shows the attack marks of predators and as such allows their identification and estimation of predation pressure. The general aim was to study predation on plasticine models of lizards and to improve methods, since the results depend on the number of plasticine models used, their spatial pattern and the duration of experiments. We estimated the density of the common wall lizard Podarcis muralis population on stone walls of a vineyard in the city of Pécs (Hungary) in August 2015 in order to imitate the real density in our experiment with plasticine models. The density of common wall lizards was 8.2 ind. /100 m2 and accordingly we placed 25 scentless plasticine lizards on the stone walls on the first transect with 10 m distance between them, which imitates the real pattern. In the second transect 25 lizard models were placed more sparsely, the distance between them being 20 m. During four weeks the predation rate was 24% in densely spaced plasticine lizards and 40% in sparsely spaced plasticine lizards, but the difference was not significant. The daily survival rate of densely spaced lizards was 0.99 (=99.1%) and that of sparsely spaced lizard models was 0.98 (=98.25%), but this difference was not significant either. On the basis of marks left on plasticine lizards, mammal predators (e.g. beech marten) dominated, while the impact of bird predators was smaller than expected. Predators attacked the head of plasticine lizards more frequently than their trunk, tail or limbs, but a significant preference of body parts was not detected. From our experience it is important to study the distribution and density of real animals, to imitate their real pattern, instead of an arbitrarily designed experiment with models. The typical scent of plasticine also could influence the results, which can be avoided by using scentless plasticine models coated with liquid rubber. We suggest the calculation of daily survival rates in order to produce results that allow the comparison of different studies

Predation of common wall lizards: experiences from a study using scentless plasticine lizards

The potential influence of predators on lacertid lizards has been studied by using models made of plasticine which shows the attack marks of predators and as such allows their identification and estimation of predation pressure. The general aim was to study predation on plasticine models of lizards and to improve methods, since the results depend on the number of plasticine models used, their spatial pattern and the duration of experiments. We estimated the density of the common wall lizard Podarcis muralis population on stone walls of a vineyard in the city of Pécs (Hungary) in August 2015 in order to imitate the real density in our experiment with plasticine models. The density of common wall lizards was 8.2 ind. /100 m2 and accordingly we placed 25 scentless plasticine lizards on the stone walls on the first transect with 10 m distance between them, which imitates the real pattern. In the second transect 25 lizard models were placed more sparsely, the distance between them being 20 m. During four weeks the predation rate was 24% in densely spaced plasticine lizards and 40% in sparsely spaced plasticine lizards, but the difference was not significant. The daily survival rate of densely spaced lizards was 0.99 (=99.1%) and that of sparsely spaced lizard models was 0.98 (=98.25%), but this difference was not significant either. On the basis of marks left on plasticine lizards, mammal predators (e.g. beech marten) dominated, while the impact of bird predators was smaller than expected. Predators attacked the head of plasticine lizards more frequently than their trunk, tail or limbs, but a significant preference of body parts was not detected. From our experience it is important to study the distribution and density of real animals, to imitate their real pattern, instead of an arbitrarily designed experiment with models. The typical scent of plasticine also could influence the results, which can be avoided by using scentless plasticine models coated with liquid rubber. We suggest the calculation of daily survival rates in order to produce results that allow the comparison of different studies

Adatok Csákányospuszta emlősfaunájához (Vértes-hegység)

Data to the mammal fauna of Csákányospuszta (Vértes Mts., Hungary). Survey of the mammal fauna was carried out on various biotopes as pine- and deciduous forest stands, spring-valley, meadows and grove sites between 9-11 June in 2017 in Csákányospuszta and its surroundings. For terrestrial species, live trapping, visual count, indirect tracks and other life signs, camera trap, while for bats, mist-netting and ultrasound-detectors were used. A total of 19 species of which 13 terrestrial and six bat species were found. Striped field mouse (Apodemus agrarius), yellow-necked mouse (A. flavicollis), wood mouse (A. sylvaticus), roe deer (Capreolus capreolus), red deer (Cervus elaphus), edible dormouse (Glis glis), brown hare (Lepus europaeus), pine marten/stone marten, (Martes martes/M. foina), Eurasian badger (Meles meles), bank vole (Myodes glareolus), wild boar (Sus scrofa), European mole (Talpa europaea) and red fox (Vulpes vulpes) were recorded as well. Among those, European mole and the edible dormouse are protected in Hungary. As for bats, western barbastelle (Barbastella barbastellus) and Bechstein’s bat (Myotis bechsteinii) as strictly protected species, further, serotine (Eptesicus serotinus), mouse-eared bat (M. myotis), noctule (Nyctalus noctula) and common pipistrelle (Pipistrellus pipistrellus) were discovered, respectively. Data series highlight the importance of effective short-term surveys in zoologically poorly known areas

Complete genomic sequencing of canine distemper virus with nanopore technology during an epizootic event

Canine distemper virus (CDV) endangers a wide range of wild animal populations, can cross species barriers and therefore representing a significant conservational and animal health risk around the globe. During spring to autumn 2021, according to our current estimates a minimum of 50 red foxes (Vulpes vulpes) died of CDV in Hungary, with CDV lesions. Oral, nasal and rectal swab samples were RT-PCR screened for Canine Distemper Virus from red fox carcasses. To investigate in more detail the origins of these CDV strains, 19 complete genomes were sequenced with a pan-genotype CDV-specific amplicon-based sequencing method developed by our laboratory and optimized for the Oxford Nanopore Technologies platform. Phylogenetic analysis of the complete genomic sequences and separately the hemagglutinin gene sequences revealed the role of the Europe lineage of CDV as a causative agent for the current epizootic. Here we highlight the growing importance of fast developing rapid sequencing technologies to aid rapid response activities during epidemics or epizootic events. We also emphasize the urgent need for improved surveillance of CDV, considering the epizootic capability of enzootic strains as reported in the current study. For such future efforts, we provide a novel NGS protocol to facilitate future genomic surveillance studies

A Single Early Introduction Governed Viral Diversity in the Second Wave of SARS-CoV-2 Epidemic in Hungary

Retrospective evaluation of past waves of the SARS-CoV-2 epidemic is key for designing optimal interventions against future waves and novel pandemics. Here we report on analysing genome sequences of SARS-CoV-2 from the first two waves of the epidemic in 2020 in Hungary, mirroring a suppression and a mitigation strategy, respectively. Our analysis reveals that the two waves markedly differed in viral diversity and transmission patterns. Specifically, unlike in several European areas or in the USA, we have found no evidence for early introduction and cryptic transmission of the virus in the first wave of the pandemic in Hungary. Despite the introduction of multiple viral lineages, extensive community spread was prevented by a timely national lockdown in March 2020. In sharp contrast, the majority of the cases in the much larger second wave can be linked to a single transmission lineage of the pan-European B.1.160 variant. This lineage was introduced unexpectedly early, followed by a two-month-long cryptic transmission before a soar of detected cases in September 2020. Epidemic analysis has revealed that the dominance of this lineage in the second wave was not associated with an intrinsic transmission advantage. This finding is further supported by the rapid replacement of B.1.160 by the alpha variant (B.1.1.7) that launched the third wave of the epidemic in February 2021. Overall, these results illustrate how the founder effect in combination with cryptic transmission, instead of repeated international introductions or higher transmissibility, can govern viral diversity

A Single Early Introduction Governed Viral Diversity in the Second Wave of SARS-CoV-2 Epidemic in Hungary

Retrospective evaluation of past waves of the SARS-CoV-2 epidemic is key for designing optimal interventions against future waves and novel pandemics. Here we report on analysing genome sequences of SARS-CoV-2 from the first two waves of the epidemic in 2020 in Hungary, mirroring a suppression and a mitigation strategy, respectively. Our analysis reveals that the two waves markedly differed in viral diversity and transmission patterns. Specifically, unlike in several European areas or in the USA, we have found no evidence for early introduction and cryptic transmission of the virus in the first wave of the pandemic in Hungary. Despite the introduction of multiple viral lineages, extensive community spread was prevented by a timely national lockdown in March 2020. In sharp contrast, the majority of the cases in the much larger second wave can be linked to a single transmission lineage of the pan-European B.1.160 variant. This lineage was introduced unexpectedly early, followed by a two-month-long cryptic transmission before a soar of detected cases in September 2020. Epidemic analysis has revealed that the dominance of this lineage in the second wave was not associated with an intrinsic transmission advantage. This finding is further supported by the rapid replacement of B.1.160 by the alpha variant (B.1.1.7) that launched the third wave of the epidemic in February 2021. Overall, these results illustrate how the founder effect in combination with cryptic transmission, instead of repeated international introductions or higher transmissibility, can govern viral diversity

In-depth Temporal Transcriptome Profiling of Monkeypox and Host Cells using Nanopore Sequencing

The recent human Monkeypox outbreak underlined the importance of studying basic biology of orthopoxviruses. However, the transcriptome of its causative agent has not been investigated before neither with short-, nor with long-read sequencing approaches. This Oxford Nanopore long-read RNA-Sequencing dataset fills this gap. It will enable the in-depth characterization of the transcriptomic architecture of the monkeypox virus, and may even make possible to annotate novel host transcripts. Moreover, our direct cDNA and native RNA sequencing reads will allow the estimation of gene expression changes of both the virus and the host cells during the infection. Overall, our study will lead to a deeper understanding of the alterations caused by the viral infection on a transcriptome level

Aedes koreicus, a vector on the rise: Pan-European genetic patterns, mitochondrial and draft genome sequencing

25openYesBackground The mosquito Aedes koreicus (Edwards, 1917) is a recent invader on the European continent that was introduced to several new places since its first detection in 2008. Compared to other exotic Aedes mosquitoes with public health significance that invaded Europe during the last decades, this species’ biology, behavior, and dispersal patterns were poorly investigated to date. Methodology/Principal findings To understand the species’ population relationships and dispersal patterns within Europe, a fragment of the cytochrome oxidase I (COI or COX1) gene was sequenced from 130 mosquitoes, collected from five countries where the species has been introduced and/or established. Oxford Nanopore and Illumina sequencing techniques were combined to generate the first complete nuclear and mitochondrial genomic sequences of Ae. koreicus from the European region. The complete genome of Ae. koreicus is 879 Mb. COI haplotype analyses identified five major groups (altogether 31 different haplotypes) and revealed a large-scale dispersal pattern between European Ae. koreicus populations. Continuous admixture of populations from Belgium, Italy, and Hungary was highlighted, additionally, haplotype diversity and clustering indicate a separation of German sequences from other populations, pointing to an independent introduction of Ae. koreicus to Europe. Finally, a genetic expansion signal was identified, suggesting the species might be present in more locations than currently detected. Conclusions/Significance Our results highlight the importance of genetic research of invasive mosquitoes to understand general dispersal patterns, reveal main dispersal routes and form the baseline of future mitigation actions. The first complete genomic sequence also provides a significant leap in the general understanding of this species, opening the possibility for future genome-related studies, such as the detection of ‘Single Nucleotide Polymorphism’ markers. Considering its public health importance, it is crucial to further investigate the species’ population genetic dynamic, including a larger sampling and additional genomic markers.Kurucz, Kornélia; Zeghbib, Safia; Arnoldi, Daniele; Marini, Giovanni; Manica, Mattia; Michelutti, Alice; Montarsi, Fabrizio; Deblauwe, Isra; Van Bortel, Wim; Smitz, Nathalie; Pfitzner, Wolf Peter; Czajka, Christina; Jöst, Artur; Kalan, Katja; Šušnjar, Jana; Ivović, Vladimir; Kuczmog, Anett; Lanszki, Zsófia; Tóth, Gábor Endre; Somogyi, Balázs A; Herczeg, Róbert; Urbán, Péter; Bueno-Marí, Rubén; Soltész, Zoltán; Kemenesi, GáborKurucz, K.; Zeghbib, S.; Arnoldi, D.; Marini, G.; Manica, M.; Michelutti, A.; Montarsi, F.; Deblauwe, I.; Van Bortel, W.; Smitz, N.; Pfitzner, W.P.; Czajka, C.; Jöst, A.; Kalan, K.; Šušnjar, J.; Ivović, V.; Kuczmog, A.; Lanszki, Z.; Tóth, G.E.; Somogyi, B.A.; Herczeg, R.; Urbán, P.; Bueno-Marí, R.; Soltész, Z.; Kemenesi, G

Isolation and genome characterization of Lloviu virus from Italian Schreibers’ bent-winged bats

13 Pág.Lloviu cuevavirus (LLOV) was the first identified member of Filoviridae family outside the Ebola and Marburgvirus genera. A massive die-off of Schreibers’ bent-winged bats (Miniopterus schreibersii) in the Iberian Peninsula in 2002 led to its discovery. Studies with recombinant and wild-type LLOV isolates confirmed the susceptibility of human-derived cell lines and primary human macrophages to LLOV infection in vitro. Based on these data, LLOV is now considered as a potential zoonotic virus with unknown pathogenicity to humans and bats. We examined bat samples from Italy for the presence of LLOV in an area outside of the currently known distribution range of the virus. We detected one positive sample from 2020, sequenced the complete coding sequence of the viral genome and established an infectious isolate of the virus. In addition, we performed the first comprehensive evolutionary analysis of the virus, using the Spanish, Hungarian and the Italian sequences. The most important achievement of this article is the establishment of an additional infectious LLOV isolate from a bat sample using the SuBK12-08 cells, demonstrating that this cell line is highly susceptible to LLOV infection. These results further confirms the role of these bats as the host of this virus, possibly throughout their entire geographic range. This is an important result to further understand the role of bats as the natural hosts for zoonotic filoviruses.This work was supported by the National Research, Development and Innovation Office, Hungary under grants NKFIH FK131465 (G.K.) and FK137778 (T.G.), and RRF-2.3.1-21-2022-00010; and the National Institutes of Health under grant R21AI169646 (E.M.). T.G. was supported by the János Bolyai Research Scholarship of the Hungarian Academy of Sciences.N