Circulation of SARS-CoV–Related Coronaviruses and Alphacoronaviruses in Bats from Croatia
Abstract
:1. Introduction
2. Materials and Methods
2.1. Ethics Statement
2.2. Bats Capturing and Sampling
2.3. Nucleic acid Extraction and RT-PCRs
2.4. Sequence and Phylogenetic Analysis
2.5. Library Construction and NGS
2.6. Data Analysis
2.7. Map to Reference Sequences
2.8. Serological Assays
3. Results
3.1. Sarbecovirus-Specific RNA Detected in Guano
3.2. The Partial RdRp Gene Sequencing Revealed the Presence of Alpha- and BetaCoV Sequences
3.3. Confirmation of Detected bat Alpha- and BetaCoVs with NGS
3.4. Positive Serology in All Bat Species Using sVNT
4. Discussion
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Calisher, C.H. Viruses in Bats. In Bats and Viruses: A New Frontier of Emerging Infectious Diseases; Wang, L.-F., Cowled, C., Eds.; John Wiley & Sons: Hoboken, NJ, USA, 2015; pp. 23–45. [Google Scholar] [CrossRef]
- Lau, S.K.; Woo, P.C.; Li, K.S.; Huang, Y.; Tsoi, H.W.; Wong, B.H.; Wong, S.S.; Leung, S.Y.; Chan, K.H.; Yuen, K.Y. Severe acute respiratory syndrome coronavirus-like virus in Chinese horseshoe bats. Proc. Natl. Acad. Sci. USA 2005, 102, 14040–14045. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Latinne, A.; Hu, B.; Olival, K.J.; Zhu, G.; Zhang, L.; Li, H.; Chmura, A.A.; Field, H.E.; Zambrana-Torrelio, C.; Epstein, J.H.; et al. Origin and cross-species transmission of bat coronaviruses in China. Nat. Commun. 2020, 11, 4235. [Google Scholar] [CrossRef] [PubMed]
- Zhou, P.; Yang, X.L.; Wang, X.G.; Hu, B.; Zhang, L.; Zhang, W.; Si, H.R.; Zhu, Y.; Li, B.; Huang, C.L.; et al. Addendum: A pneumonia outbreak associated with a new coronavirus of probable bat origin. Nature 2020, 588, E6. [Google Scholar] [CrossRef]
- Temmam, S.; Vongphayloth, K.; Baquero, E.; Munier, S.; Bonomi, M.; Regnault, B.; Douangboubpha, B.; Karami, Y.; Chrétien, D.; Sanamxay, D.; et al. Bat coronaviruses related to SARS-CoV-2 and infectious for human cells. Nature 2022, 604, 330–336. [Google Scholar] [CrossRef]
- Guan, Y.; Zheng, B.J.; He, Y.Q.; Liu, X.L.; Zhuang, Z.X.; Cheung, C.L.; Luo, S.W.; Li, P.H.; Zhang, L.J.; Guan, Y.J.; et al. Isolation and characterization of viruses related to the SARS coronavirus from animals in southern China. Science 2003, 302, 276–278. [Google Scholar] [CrossRef] [Green Version]
- He, B.; Zhang, Y.; Xu, L.; Yang, W.; Yang, F.; Feng, Y.; Xia, L.; Zhou, J.; Zhen, W.; Feng, Y.; et al. Identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in China. J. Virol. 2014, 88, 7070–7082. [Google Scholar] [CrossRef] [Green Version]
- Lin, X.D.; Wang, W.; Hao, Z.Y.; Wang, Z.X.; Guo, W.P.; Guan, X.Q.; Wang, M.R.; Wang, H.W.; Zhou, R.H.; Li, M.H.; et al. Extensive diversity of coronaviruses in bats from China. Virology 2017, 507, 1–10. [Google Scholar] [CrossRef] [PubMed]
- Wang, L.; Fu, S.; Cao, Y.; Zhang, H.; Feng, Y.; Yang, W.; Nie, K.; Ma, X.; Liang, G. Discovery and genetic analysis of novel coronaviruses in least horseshoe bats in southwestern China. Emerg. Microbes Infect. 2017, 6, e14. [Google Scholar] [CrossRef] [Green Version]
- Guo, H.; Hu, B.; Si, H.R.; Zhu, Y.; Zhang, W.; Li, B.; Li, A.; Geng, R.; Lin, H.F.; Yang, X.L.; et al. Identification of a novel lineage bat SARS-related coronaviruses that use bat ACE2 receptor. Emerg. Microbes Infect. 2021, 10, 1507–1514. [Google Scholar] [CrossRef]
- Murakami, S.; Kitamura, T.; Matsugo, H.; Kamiki, H.; Oyabu, K.; Sekine, W.; Takenaka-Uema, A.; Sakai-Tagawa, Y.; Kawaoka, Y.; Horimoto, T. Isolation of Bat Sarbecoviruses, Japan. Emerg. Infect. Dis. 2022, 28, 2500–2503. [Google Scholar] [CrossRef]
- Murakami, S.; Kitamura, T.; Suzuki, J.; Sato, R.; Aoi, T.; Fujii, M.; Matsugo, H.; Kamiki, H.; Ishida, H.; Takenaka-Uema, A.; et al. Detection and Characterization of Bat Sarbecovirus Phylogenetically Related to SARS-CoV-2, Japan. Emerg. Infect. Dis. 2020, 26, 3025–3029. [Google Scholar] [CrossRef] [PubMed]
- Mallapaty, S. Coronaviruses closely related to the pandemic virus discovered in Japan and Cambodia. Nature 2020, 588, 15–16. [Google Scholar] [CrossRef] [PubMed]
- Wacharapluesadee, S.; Tan, C.W.; Maneeorn, P.; Duengkae, P.; Zhu, F.; Joyjinda, Y.; Kaewpom, T.; Chia, W.N.; Ampoot, W.; Lim, B.L.; et al. Evidence for SARS-CoV-2 related coronaviruses circulating in bats and pangolins in Southeast Asia. Nat. Commun. 2021, 12, 972. [Google Scholar] [CrossRef] [PubMed]
- Wells, H.L.; Letko, M.; Lasso, G.; Ssebide, B.; Nziza, J.; Byarugaba, D.K.; Navarrete-Macias, I.; Liang, E.; Cranfield, M.; Han, B.A.; et al. The evolutionary history of ACE2 usage within the coronavirus subgenus Sarbecovirus. Virus Evol. 2021, 7, veab007. [Google Scholar] [CrossRef]
- Delaune, D.; Hul, V.; Karlsson, E.A.; Hassanin, A.; Ou, T.P.; Baidaliuk, A.; Gámbaro, F.; Prot, M.; Tu, V.T.; Chea, S.; et al. A novel SARS-CoV-2 related coronavirus in bats from Cambodia. Nat. Commun. 2021, 12, 6563. [Google Scholar] [CrossRef]
- Seifert, S.N.; Bai, S.; Fawcett, S.; Norton, E.B.; Zwezdaryk, K.J.; Robinson, J.; Gunn, B.; Letko, M. An ACE2-dependent Sarbecovirus in Russian bats is resistant to SARS-CoV-2 vaccines. PLoS Pathog. 2022, 18, e1010828. [Google Scholar] [CrossRef] [PubMed]
- Rihtarič, D.; Hostnik, P.; Steyer, P.; Grom, J.; Toplak, I. Identification of SARS-like coronaviruses in horseshoe bats (Rhinolophus hipposideros) in Slovenia. Arch. Virol. 2010, 155, 507–514. [Google Scholar] [CrossRef]
- Drexler, J.F.; Gloza-Rausch, F.; Glende, J.; Corman, V.M.; Muth, D.; Goettsche, M.; Seebens, A.; Niedrig, M.; Pfefferle, S.; Yordanov, S.; et al. Genomic characterization of severe acute respiratory syndrome-related coronavirus in European bats and classification of coronaviruses based on partial RNA-dependent RNA polymerase gene sequences. J. Virol. 2010, 84, 11336–11349. [Google Scholar] [CrossRef] [Green Version]
- Balboni, A.; Palladini, A.; Bogliani, G.; Battiliani, M. Detection of a virus related to betacoronaviruses in Italian greater horseshoe bats. Epidemiol. Infect. 2011, 139, 216–219. [Google Scholar] [CrossRef] [Green Version]
- Kemenesi, G.; Dallos, B.; Görföl, T.; Boldoglh, S.; Estók, P.; Kurucz, K.; Kutas, A.; Földes, F.; Oldal, M.; Németh, V.; et al. Molecular survey of RNA viruses in Hungarian bats: Discovering novel astroviruses, coronaviruses, and caliciviruses. Vector Borne Zoonotic Dis. 2014, 14, 846–855. [Google Scholar] [CrossRef]
- Crook, J.M.; Murphy, I.; Carter, D.P.; Pullan, S.T.; Carroll, M.; Vipond, R.; Cunningham, A.A.; Bell, D. Metagenomic identification of a new sarbecovirus from horseshoe bats in Europe. Sci. Rep. 2021, 11, 14723. [Google Scholar] [CrossRef] [PubMed]
- Orłowska, A.; Smreczak, M.; Thor, K.; Niedbalska, M.; Pawelec, D.; Trebas, P.; Rola, J. The Genetic Characterization of the First Detected Bat Coronaviruses in Poland Revealed SARS-Related Types and Alphacoronaviruses. Viruses 2022, 14, 1914. [Google Scholar] [CrossRef]
- WOAH. SARS-CoV-2 in Animals—Situation Report 19. 2022. Available online: https://www.woah.org/app/uploads/2022/12/sars-cov-2-situation-report-19.pdf (accessed on 9 February 2023).
- Oreshkova, N.; Molenaar, R.J.; Vreman, S.; Harders, F.; Oude Munnink, B.B.; Hakze-van der Honing, R.W.; Gerhards, N.; Tolsma, P.; Bouwstra, R.; Sikkema, R.S.; et al. SARS-CoV-2 infection in farmed minks, the Netherlands, April and May 2020. Euro. Surveill 2020, 25, 2001005. [Google Scholar] [CrossRef]
- Oude Munnink, B.B.; Sikkema, R.S.; Nieuwenhuijse, D.F.; Molenaar, R.J.; Munger, E.; Molenkamp, R.; van der Spek, A.; Tolsma, P.; Rietveld, A.; Brouwer, M.; et al. Transmission of SARS-CoV-2 on mink farms between humans and mink and back to humans. Science 2021, 371, 172–177. [Google Scholar] [CrossRef] [PubMed]
- Yen, H.L.; Sit, T.H.C.; Brackman, C.J.; Chuk, S.S.Y.; Gu, H.; Tam, K.W.S.; Law, P.Y.T.; Leung, G.M.; Peiris, M.; Poon, L.L.M.; et al. Transmission of SARS-CoV-2 delta variant (AY.127) from pet hamsters to humans, leading to onward human-to-human transmission: A case study. Lancet 2022, 399, 1070–1078. [Google Scholar] [CrossRef] [PubMed]
- Martins, M.; Boggiatto, P.M.; Buckley, A.; Cassmann, E.D.; Falkenberg, S.; Caserta, L.C.; Fernandes, M.H.V.; Kanipe, C.; Lager, K.; Palmer, M.V. From Deer-to-Deer: SARS-CoV-2 is efficiently transmitted and presents broad tissue tropism and replication sites in white-tailed deer. PLoS Pathog. 2022, 18, e1010197. [Google Scholar] [CrossRef]
- Sila, T.; Sunghan, J.; Laochareonsuk, W.; Surasombatpattana, S.; Kongkamol, C.; Ingviya, T.; Siripaitoon, P.; Kositpantawong, N.; Kanchanasuwan, S.; Hortiwakul, T.; et al. Suspected Cat-to-Human Transmission of SARS-CoV-2, Thailand, July-September 2021. Emerg. Infect. Dis. 2022, 28, 1485–1488. [Google Scholar] [CrossRef]
- Schlottau, K.; Rissmann, M.; Graaf, A.; Schön, J.; Sehl, J.; Wylezich, C.; Höper, D.; Mettenleiter, T.C.; Balkema-Buschmann, A.; Harder, T.; et al. SARS-CoV-2 in fruit bats, ferrets, pigs, and chickens: An experimental transmission study. Lancet Microbe 2020, 1, e218–e225. [Google Scholar] [CrossRef]
- Hamidović, D.; Josić, D.; Kipson, M.; Komerički, A.; Pintar, V.; Rnjak, D.; Rnjak, G.; Zrnčić, V.; Zadravec, M.; Žvorc, P.; et al. The first assessment of the state of conservation of bats—Chiroptera in Croatia. 2019; unpublished. [Google Scholar]
- Ministry of Environmental and Nature Protection of the Republic of Croatia, Nature Protection Directorate & State Institute for Nature Protection. Inf.EUROBATS.MoP7.12—Sixth National Report on the Implementation of the Agreement; Ministry of Environmental and Nature Protection of the Republic of Croatia, Nature Protection Directorate & State Institute for Nature Protection: Zagreb, Croatia, 2014. [Google Scholar]
- Tvrtković, N. The findings of Mehely’s horseshoe bat (Chiroptera) in Croatia in the last century were mistakes in identification. Nat. Croat 2016, 25, 165–172. [Google Scholar] [CrossRef]
- Pavlinić, I.; Đaković, M.; Tvrtković, N. The atlas of Croatian bats (Chiroptera) part I. Nat. Croat 2010, 19, 295–337. [Google Scholar]
- Gazaryan, S.; Bücs, S.; Çoraman, E. Miniopterus schreibersii (Errata Version Published in 2021). IUCN Red List. Threat. Species 2020, e.T81633057A195856522. [Google Scholar] [CrossRef]
- Wright, P.; Newton, J.; Agnelli, P.; Budinski, I.; Di Salvo, I.; Flaquer, C.; Fulco, A.; Georgiakakis, P.; Martinoli, A.; Mas, M.; et al. Hydrogen isotopes reveal evidence of migration of Miniopterus schreibersii in Europe. BMC Ecol. 2020, 20, 52. [Google Scholar] [CrossRef]
- Hutterer, R.; Ivanova, T.; Meyer-Cords, C.; Rodrigues, L. Bat Migrations in Europe: A Review of Banding Data and Literature; Federal Agency for Nature Conservation in Germany: Bonn, Germany, 2005; Volume 28, p. 162. [Google Scholar]
- Šimić, I.; Lojkić, I.; Krešić, N.; Cliquet, F.; Picard-Meyer, E.; Wasniewski, M.; Ćukušić, A.; Zrnčić, V.; Bedeković, T. Molecular and serological survey of lyssaviruses in Croatian bat populations. BMC Vet. Res. 2018, 14, 274. [Google Scholar] [CrossRef]
- Šimić, I.; Zorec, T.M.; Lojkić, I.; Krešić, N.; Poljak, M.; Cliquet, F.; Picard-Meyer, E.; Wasniewski, M.; Zrnčić, V.; Ćukušić, A.; et al. Viral Metagenomic Profiling of Croatian Bat Population Reveals Sample and Habitat Dependent Diversity. Viruses 2020, 12, 891. [Google Scholar] [CrossRef]
- IUCN SSC Bat Specialist Group (BSG). Recommended Strategy for Researchers to Reduce the Risk of Transmission of SARS-CoV-2 from Humans to Bats. MAP: Minimize, Assess, Protect. Available online: https://www.iucnbsg.org/uploads/6/5/0/9/6509077/map_recommendations_for_researchers_v._1.0_final.pdf (accessed on 23 September 2020).
- IUCN SSC Wildlife Health Specialist Group. Guidelines for Working with Free-Ranging Wild Mammals in the Era of the COVID-19 Pandemic. Available online: http://www.iucn-whsg.org/sites/default/files/En_WHSG%20and%20OIE%20COVID-19%20Guidelines_0.pdf (accessed on 23 September 2020).
- Corman, V.M.; Landt, O.; Kaiser, M.; Molenkamp, R.; Meijer, A.; Chu, D.K.W.; Bleicker, T.; Brünink, S.; Schneider, J.; Schmidt, M.L.; et al. Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR. Euro. Surveill 2020, 25, 2000045. [Google Scholar] [CrossRef] [Green Version]
- Lu, X.; Wang, L.; Sakthivel, S.K.; Whitaker, B.; Murray, J.; Kamili, S.; Lynch, B.; Malapati, L.; Burke, S.A.; Harcourt, J.; et al. US CDC Real-Time Reverse Transcription PCR Panel for Detection of Severe Acute Respiratory Syndrome Coronavirus 2. Emerg. Infect. Dis. 2020, 26, 1654–1665. [Google Scholar] [CrossRef]
- Xiu, L.; Binder, R.A.; Alarja, N.A.; Kochek, K.; Coleman, K.K.; Than, S.T.; Bailey, E.S.; Bui, V.N.; Toh, T.H.; Erdman, D.D.; et al. A RT-PCR assay for the detection of coronaviruses from four genera. J. Clin. Virol. 2020, 128, 104391. [Google Scholar] [CrossRef] [PubMed]
- Toussaint, J.F.; Sailleau, C.; Breard, E.; Zientara, S.; De Clercq, K. Bluetongue virus detection by two real-time RT-qPCRs targeting two different genomic segments. J. Virol. Methods 2007, 140, 115–123. [Google Scholar] [CrossRef] [PubMed]
- Tamura, K.; Stecher, G.; and Kumar, S. MEGA 11: Molecular Evolutionary Genetics Analysis Version 11. Mol. Biol. Evol. 2021, 38, 3022–3027. [Google Scholar] [CrossRef] [PubMed]
- Bolger, A.M.; Lohse, M.; Usadel, B. Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 2014, 30, 2114–2120. [Google Scholar] [CrossRef] [Green Version]
- Wood, D.E.; Lu, J.; Langmead, B. Improved metagenomic analysis with Kraken 2. Genome Biol. 2019, 20, 257. [Google Scholar] [CrossRef] [Green Version]
- Lu, J.; Breitwieser, F.P.; Thielen, P.; Salzberg, S.L. Bracken: Estimating species abundance in metagenomics data. PeerJ Comput. Sci. 2017, 3, e104. [Google Scholar] [CrossRef] [Green Version]
- Prjibelski, A.; Antipov, D.; Meleshko, D.; Lapidus, A.; Korobeynikov, A. Using SPAdes De Novo Assembler. Curr. Protoc. Bioinform. 2020, 70, e102. [Google Scholar] [CrossRef]
- Mikheenko, A.; Prjibelski, A.; Saveliev, V.; Antipov, D.; Gurevich, A. Versatile genome assembly evaluation with QUAST-LG. Bioinformatics 2018, 34, i142–i150. [Google Scholar] [CrossRef]
- Mölder, F.; Jablonski, K.P.; Letcher, B.; Hall, M.B.; Tomkins-Tinch, C.H.; Sochat, V.; Forster, J.; Lee, S.; Twardziok, S.O.; Kanitz, A.; et al. Sustainable data analysis with Snakemake. F1000Research 2021, 10, 33. [Google Scholar] [CrossRef] [PubMed]
- Picard-Meyer, E.; Servat, A.; Wasniewski, M.; Gaillard, M.; Borel, C.; Cliquet, F. Bat rabies surveillance in France: First report of unusual mortality among serotine bats. BMC Vet. Res. 2017, 13, 387. [Google Scholar] [CrossRef]
- Jemeršić, L.; Lojkić, I.; Krešić, N.; Keros, T.; Zelenika, T.A.; Jurinović, L.; Skok, D.; Bata, I.; Boras, J.; Habrun, B.; et al. Investigating the Presence of SARS-CoV-2 in Free-Living and Captive Animals. Pathogens 2021, 10, 635. [Google Scholar] [CrossRef] [PubMed]
- Peel, A.J.; Wells, K.; Giles, J.; Boyd, V.; Burroughs, A.; Edson, D.; Crameri, G.; Baker, M.L.; Field, H.; Wang, L.F.; et al. Synchronous shedding of multiple bat paramyxoviruses coincides with peak periods of Hendra virus spillover. Emerg. Microbes Infect. 2019, 8, 1314–1323. [Google Scholar] [CrossRef] [Green Version]
- Embregts, C.W.E.; Verstrepen, B.; Langermans, J.A.M.; Böszörményi, K.P.; Sikkema, R.S.; de Vries, R.D.; Hoffmann, D.; Wernike, K.; Smit, L.A.M.; Zhao, S.; et al. Evaluation of a multi-species SARS-CoV-2 surrogate virus neutralization test. One Health 2021, 13, 100313. [Google Scholar] [CrossRef] [PubMed]
- Tan, C.W.; Chia, W.N.; Qin, X.; Liu, P.; Chen, M.I.; Tiu, C.; Hu, Z.; Chen, V.C.; Young, B.E.; Sia, W.R.; et al. A SARS-CoV-2 surrogate virus neutralization test based on antibody-mediated blockage of ACE2–spike protein–protein interaction. Nat. Biotechnol. 2020, 38, 1073–1078. [Google Scholar] [CrossRef]
- Perera, R.A.P.M.; Ko, R.; Tsang, O.T.Y.; Hui, D.S.C.; Kwan, M.Y.M.; Brackman, C.J.; To, E.M.W.; Yen, H.L.; Leung, K.; Cheng, S.M.S.; et al. Evaluation of a SARS-CoV-2 Surrogate Virus Neutralization Test for Detection of Antibody in Human, Canine, Cat, and Hamster Sera. J. Clin. Microbiol. 2021, 59, e02504-20. [Google Scholar] [CrossRef] [PubMed]
- Hofmann, N.; Grossegesse, M.; Neumann, M.; Schaade, L.; Nitsche, A. Evaluation of a commercial ELISA as alternative to plaque reduction neutralization test to detect neutralizing antibodies against SARS-CoV-2. Sci. Rep. 2022, 12, 3549. [Google Scholar] [CrossRef] [PubMed]
- Willoughby, A.R.; Phelps, K.L.; PREDICT Consortium; Olival, K.J. A Comparative Analysis of Viral Richness and Viral Sharing in Cave-Roosting Bats. Diversity 2017, 9, 35. [Google Scholar] [CrossRef] [Green Version]
N | E-gene Real-Time RT-PCR | Pan-CoV RdRp RT-PCR | |
---|---|---|---|
Bat droppings | 11 | 0 | 0 |
Guano | 19 | 5 (26.31%) | 3 (15.78%) |
Bat Species | Number of Tested Bats | sVNT Positive Results |
---|---|---|
M. schreibersii | 13 | 4 (30.76%) |
M. capaccinii | 10 | 3 (30.00%) |
M. myotis | 8 | 3 (37.50%) |
R. ferrumequinum | 7 | 1 (14.28%) |
TOTAL | 38 | 11 (28.94%) |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2023 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Brnić, D.; Lojkić, I.; Krešić, N.; Zrnčić, V.; Ružanović, L.; Mikuletič, T.; Bosilj, M.; Steyer, A.; Keros, T.; Habrun, B.; et al. Circulation of SARS-CoV–Related Coronaviruses and Alphacoronaviruses in Bats from Croatia. Microorganisms 2023, 11, 959. https://doi.org/10.3390/microorganisms11040959
Brnić D, Lojkić I, Krešić N, Zrnčić V, Ružanović L, Mikuletič T, Bosilj M, Steyer A, Keros T, Habrun B, et al. Circulation of SARS-CoV–Related Coronaviruses and Alphacoronaviruses in Bats from Croatia. Microorganisms. 2023; 11(4):959. https://doi.org/10.3390/microorganisms11040959
Chicago/Turabian StyleBrnić, Dragan, Ivana Lojkić, Nina Krešić, Vida Zrnčić, Lea Ružanović, Tina Mikuletič, Martin Bosilj, Andrej Steyer, Tomislav Keros, Boris Habrun, and et al. 2023. "Circulation of SARS-CoV–Related Coronaviruses and Alphacoronaviruses in Bats from Croatia" Microorganisms 11, no. 4: 959. https://doi.org/10.3390/microorganisms11040959