Epidemiology and Evolution of Bovine Viral Diarrhea Virus (BVDV) in Uruguay: A 10-Year Study
Abstract
1. Introduction
2. Materials and Methods
2.1. Sample Collection
2.2. RNA Extraction, Reverse Transcription and Sample Screening by Real-Time PCR
2.3. Amplification by Conventional PCR
2.4. Phylogenetic Analysis
2.5. Population Dynamics Analysis of Uruguayan BVDV-2b
3. Results
3.1. Genetic Diversity of Uruguayan BVDV Strains
3.2. Population Dynamics Analysis of Uruguayan BVDV-2b Strains
4. Discussion
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
- Baker, J.C. Bovine viral diarrhea virus: A review. J. Am. Vet. Med. Assoc. 1987, 190, 1449–1458. [Google Scholar] [CrossRef] [PubMed]
- Baker, J.C. The clinical manifestations of bovine viral diarrhea infection. Vet. Clin. N. Am. Food Anim. Pract. 1995, 11, 425–445. [Google Scholar] [CrossRef]
- Houe, H. Epidemiological features and economical importance of bovine virus diarrhoea virus (BVDV) infections. Vet. Microbiol. 1999, 64, 89–107, Erratum in: Vet. Microbiol. 2003, 93, 275–276. [Google Scholar] [CrossRef]
- Meyers, G.; Thiel, H.J. Molecular characterization of pestiviruses. Adv. Virus Res. 1996, 47, 53–118. [Google Scholar] [CrossRef]
- International Committee on Taxonomy of Viruses ICTV. Available online: https://talk.ictvonline.org/ (accessed on 25 August 2025).
- Vilcek, S.; Paton, D.J.; Durkovic, B.; Strojny, L.; Ibata, G.; Moussa, A.; Loitsch, A.; Rossmanith, W.; Vega, S.; Scicluna, M.T.; et al. Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups. Arch. Virol. 2001, 146, 99–115. [Google Scholar] [CrossRef]
- Jackova, A.; Novackova, M.; Pelletier, C.; Audeval, C.; Gueneau, E.; Haffar, A.; Petit, E.; Rehby, L.; Vilcek, S. The extended genetic diversity of BVDV-1: Typing of BVDV isolates from France. Vet. Res. Commun. 2008, 32, 7–11. [Google Scholar] [CrossRef]
- Nagai, M.; Hayashi, M.; Itou, M.; Fukutomi, T.; Akashi, H.; Kida, H.; Sakoda, Y. Identification of new genetic subtypes of bovine viral diarrhea virus genotype 1 isolated in Japan. Virus Genes 2008, 36, 135–139. [Google Scholar] [CrossRef]
- Xue, F.; Zhu, Y.M.; Li, J.; Zhu, L.C.; Ren, X.G.; Feng, J.K.; Shi, H.F.; Gao, Y.R. Genotyping of bovine viral diarrhea viruses from cattle in China between 2005 and 2008. Vet. Microbiol. 2010, 143, 379–383. [Google Scholar] [CrossRef] [PubMed]
- Luzzago, C.; Lauzi, S.; Ebranati, E.; Giammarioli, M.; Moreno, A.; Cannella, V.; Masoero, L.; Canelli, E.; Guercio, A.; Caruso, C.; et al. Extended genetic diversity of bovine viral diarrhea virus and frequency of genotypes and subtypes in cattle in Italy between 1995 and 2013. BioMed Res. Int. 2014, 2014, 147145. [Google Scholar] [CrossRef] [PubMed]
- Giammarioli, M.; Ceglie, L.; Rossi, E.; Bazzucchi, M.; Casciari, C.; Petrini, S.; De Mia, G.M. Increased genetic diversity of BVDV-1: Recent findings and implications thereof. Virus Genes 2015, 50, 147–151. [Google Scholar] [CrossRef] [PubMed]
- Deng, M.; Ji, S.; Fei, W.; Raza, S.; He, C.; Chen, Y.; Chen, H.; Guo, A. Prevalence study and genetic typing of bovine viral diarrhea virus (BVDV) in four bovine species in China. PLoS ONE 2015, 10, e0121718, Erratum in: PLoS ONE 2015, 10, e0134777. https://doi.org/10.1371/journal.pone.0134777. [Google Scholar] [CrossRef]
- Deng, M.; Chen, N.; Guidarini, C.; Xu, Z.; Zhang, J.; Cai, L.; Yuan, S.; Sun, Y.; Metcalfe, L. Prevalence and genetic diversity of bovine viral diarrhea virus in dairy herds of China. Vet. Microbiol. 2020, 242, 108565. [Google Scholar] [CrossRef]
- Vilcek, S.; Herring, A.J.; Herring, J.A.; Nettleton, P.F.; Lowings, J.P.; Paton, D.J. Pestiviruses isolated from pigs, cattle and sheep can be allocated into at least three genogroups using polymerase chain reaction and restriction endonuclease analysis. Arch. Virol. 1994, 136, 309–323. [Google Scholar] [CrossRef]
- Flores, E.F.; Ridpath, J.F.; Weiblen, R.; Vogel, F.S.; Gil, L.H. Phylogenetic analysis of Brazilian bovine viral diarrhea virus type 2 (BVDV-2) isolates: Evidence for a subgenotype within BVDV-2. Virus Res. 2002, 87, 51–60. [Google Scholar] [CrossRef] [PubMed]
- Peterhans, E.; Bachofen, C.; Stalder, H.; Schweizer, M. Cytopathic bovine viral diarrhea viruses (BVDV): Emerging pestiviruses doomed to extinction. Vet. Res. 2010, 41, 44. [Google Scholar] [CrossRef]
- Jenckel, M.; Höper, D.; Schirrmeier, H.; Reimann, I.; Goller, K.V.; Hoffmann, B.; Beer, M. Mixed triple: Allied viruses in unique recent isolates of highly virulent type 2 bovine viral diarrhea virus detected by deep sequencing. J. Virol. 2014, 88, 6983–6992. [Google Scholar] [CrossRef]
- de Oliveira, P.S.B.; Júnior, J.V.J.S.; Weiblen, R.; Flores, E.F. A new (old) bovine viral diarrhea virus 2 subtype: BVDV-2e. Arch. Virol. 2022, 167, 2545–2553. [Google Scholar] [CrossRef] [PubMed]
- Mishra, N.; Rajukumar, K.; Pateriya, A.; Kumar, M.; Dubey, P.; Behera, S.; Verma, A.; Bhardwaj, P.; Kulkarni, D.; Vijaykrishna, D.; et al. Identification and molecular characterization of novel and divergent HoBi-like pestiviruses from naturally infected cattle in India. Vet. Microbiol. 2014, 174, 239–246. [Google Scholar] [CrossRef]
- Silveira, S.; Cibulski, S.P.; Junqueira, D.M.; Mósena, A.C.S.; Weber, M.N.; Mayer, F.Q.; Canal, C.W. Phylogenetic and evolutionary analysis of HoBi-like pestivirus: Insights into origin and dispersal. Transbound. Emerg. Dis. 2020, 67, 1909–1917. [Google Scholar] [CrossRef] [PubMed]
- Kalaiyarasu, S.; Mishra, N.; Jayalakshmi, K.; Selvaraj, P.; Sudhakar, S.B.; Jhade, S.K.; Sood, R.; Premalatha, N.; Singh, V.P. Molecular characterization of recent HoBi-like pestivirus isolates from cattle showing mucosal disease-like signs in India reveals emergence of a novel genetic lineage. Transbound. Emerg. Dis. 2022, 69, 308–326. [Google Scholar] [CrossRef]
- Spetter, M.J.; Uriarte, E.L.L.; Verna, A.E.; Odeón, A.C.; Altamiranda, E.A.G. Temporal and geographic dynamics of bovine viral diarrhea virus in American countries. Res. Vet. Sci. 2022, 153, 66–73. [Google Scholar] [CrossRef]
- Bauermann, F.V.; Ridpath, J.F.; Weiblen, R.; Flores, E.F. HoBi-like viruses: An emerging group of pestiviruses. J. Vet. Diagn. Investig. 2013, 25, 6–15. [Google Scholar] [CrossRef]
- Ridpath, J.F. Bovine viral diarrhea virus: Global status. Vet. Clin. N. Am. Food Anim. Pract. 2010, 26, 105–121. [Google Scholar] [CrossRef] [PubMed]
- Ministerio de Ganadería Agricultura y Pesca-Uruguay (MGAP)-Dirección de Estadística Agropecuaria (DIEA). Available online: https://www.gub.uy/ministerio-ganaderia-agricultura-pesca/diea/anuario2024 (accessed on 25 August 2025).
- Saizar, J.; Gil, A. Estudio serológico de la Diarrea Viral Bovina en bovinos en el Uruguay. Vet. (Montev.) 1998, 34, 9–14. [Google Scholar]
- Guarino, H.; Núñez, A.; Repiso, M.; Gil, A.; Dargatz, D. Prevalence of serum antibodies to bovine herpesvirus-1 and bovine viral diarrhea virus in beef cattle in Uruguay. Prev. Vet. Med. 2008, 85, 34–40. [Google Scholar] [CrossRef] [PubMed]
- Yarnall, M.J.; Thrusfield, M.V. Engaging veterinarians and farmers in eradicating bovine viral diarrhoea: A systematic review of economic impact. Vet. Rec. 2017, 181, 347. [Google Scholar] [CrossRef]
- PLANISA_Presidencia de la República Oriental del Uruguay-Plan Nacional de Investigación en Sanidad Animal-2009. Available online: http://archivo.presidencia.gub.uy/_web/MEM_2009/MGAP.pdf (accessed on 25 August 2025).
- Maya, L.; Puentes, R.; Reolón, E.; Acuña, P.; Riet, F.; Rivero, R.; Cristina, J.; Colina, R. Molecular diversity of bovine viral diarrhea virus in uruguay. Arch. Virol. 2016, 161, 529–535. [Google Scholar] [CrossRef]
- Maya, L.; Macías-Rioseco, M.; Silveira, C.; Giannitti, F.; Castells, M.; Salvo, M.; Rivero, R.; Cristina, J.; Gianneechini, E.; Puentes, R.; et al. An extensive field study reveals the circulation of new genetic variants of subtype 1a of bovine viral diarrhea virus in Uruguay. Arch. Virol. 2020, 165, 145–156. [Google Scholar] [CrossRef]
- Maya, L.; Panzera, Y.; Pérez, R.; Marandino, A.; Colina, R. Coding-complete genome sequences of two bovine viral diarrhea virus 1a isolates from Uruguay. Microbiol. Resour. Announc. 2024, 13, e0091723. [Google Scholar] [CrossRef]
- Kumar, S.; Stecher, G.; Li, M.; Knyaz, C.; Tamura, K. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol. Biol. Evol. 2018, 35, 1547–1549. [Google Scholar] [CrossRef]
- Posada, D. jModelTest: Phylogenetic model averaging. Mol. Biol. Evol. 2008, 25, 1253–1256. [Google Scholar] [CrossRef] [PubMed]
- Rambaut, A.; Lam, T.T.; Max Carvalho, L.; Pybus, O.G. Exploring the temporal structure of heterochronous sequences using TempEst (formerly Path-O-Gen). Virus Evol. 2016, 2, vew007. [Google Scholar] [CrossRef]
- Suchard, M.A.; Lemey, P.; Baele, G.; Ayres, D.L.; Drummond, A.J.; Rambaut, A. Bayesian phylogenetic and phylodynamic data integration using BEAST 1.10. Virus Evol. 2018, 4, vey016. [Google Scholar] [CrossRef]
- Drummond, A.J.; Rambaut, A.; Shapiro, B.; Pybus, O.G. Bayesian coalescent inference of past population dynamics from molecular sequences. Mol. Biol. Evol. 2005, 22, 1185–1192. [Google Scholar] [CrossRef] [PubMed]
- Lemey, P.; Rambaut, A.; Drummond, A.J.; Suchard, M.A. Bayesian phylogeography finds its roots. PLoS Comput. Biol. 2009, 5, e1000520. [Google Scholar] [CrossRef]
- Reichel, M.P.; Lanyon, S.R.; Hill, F.I. Perspectives on Current Challenges and Opportunities for Bovine Viral Diarrhoea Virus Eradication in Australia and New Zealand. Pathogens 2018, 7, 14. [Google Scholar] [CrossRef]
- Oguejiofor, C.F.; Thomas, C.; Cheng, Z.; Wathes, D.C. Mechanisms linking bovine viral diarrhea virus (BVDV) infection with infertility in cattle. Anim. Health Res. Rev. 2019, 20, 72–85. [Google Scholar] [CrossRef]
- Bianchi, E.; Martins, M.; Weiblen, R.; Flores, E.F. Perfil genotípico e antigênico de amostras do vírus da diarréia viral bovina isoladas no Rio Grande do Sul (2000–2010). Pesqui. Vet. Bras. 2011, 31, 649–655. [Google Scholar] [CrossRef]
- Weber, M.; Silveira, S.; Machado, G.; Groff, F.; Mósena, A.; Budaszewski, R.; Dupont, P.; Corbellini, L.; Canal, C. High frequency of bovine viral diarrhea virus type 2 in Southern Brazil. Virus Res. 2014, 191, 117–124. [Google Scholar] [CrossRef]
- Dias, R.K.; Cargnelutti, J.F.; Weber, M.N.; Canal, C.W.; Bauermann, F.V.; Ridpath, J.F.; Weiblen, R.; Flores, E.F. Antigenic diversity of Brazilian isolates of HoBi-like pestiviruses. Vet. Microbiol. 2017, 203, 221–228. [Google Scholar] [CrossRef] [PubMed]
- Pecora, A.; Aguirreburualde, M.S.P.; Malacari, D.A.; Zabal, O.; Sala, J.M.; Konrad, J.L.; Caspe, S.G.; Bauermann, F.; Ridpath, J.; Santos, M.J.D. Serologic evidence of HoBi-like virus circulation in Argentinean water buffalo. J. Vet. Diagn. Investig. 2017, 29, 926–929. [Google Scholar] [CrossRef]
- Silveira, S.; Weber, M.N.; Mósena, A.C.S.; da Silva, M.S.; Streck, A.F.; Pescador, C.A.; Flores, E.F.; Weiblen, R.; Driemeier, D.; Ridpath, J.F.; et al. Genetic Diversity of Brazilian Bovine Pestiviruses Detected Between 1995 and 2014. Transbound. Emerg. Dis. 2017, 64, 613–623. [Google Scholar] [CrossRef] [PubMed]
- Monteiro, F.L.; Martins, B.; Cargnelutti, J.F.; Noll, J.G.; Weiblen, R.; Flores, E.F. Genetic identification of pestiviruses from beef cattle in Southern Brazil. Braz. J. Microbiol. 2019, 50, 557–563. [Google Scholar] [CrossRef]
- Margineda, C.A.; Ferreyra, F.M.; Masnyj, F.; Audrito, M.; Favaro, P.M.; María José, D.S.; Pecora, A. HoBi-like pestivirus in 2 cases of fatal respiratory disease of feedlot cattle in Argentina. J. Vet. Diagn. Investig. 2022, 34, 693–698. [Google Scholar] [CrossRef]
- Mosena, A.C.S.; Wolf, J.M.; Paim, W.P.; Baumbach, L.F.; da Silva, M.S.; Silveira, S.; Olegário, J.D.C.; Budaszewski, R.D.F.; Weber, M.N.; Canal, C.W. Temporal analysis of bovine pestivirus diversity in Brazil. Braz. J. Microbiol. 2022, 53, 1675–1682. [Google Scholar] [CrossRef]
- Strong, R.; Errington, J.; Cook, R.; Ross-Smith, N.; Wakeley, P.; Steinbach, F. Increased phylogenetic diversity of bovine viral diarrhoea virus type 1 isolates in England and Wales since 2001. Vet. Microbiol. 2013, 162, 315–320. [Google Scholar] [CrossRef]
- Mósena, A.C.; Weber, M.N.; Cibulski, S.P.; Silveira, S.; Silva, M.S.; Mayer, F.Q.; Canal, C.W. Genomic characterization of a bovine viral diarrhea virus subtype 1i in Brazil. Arch. Virol. 2017, 162, 1119–1123. [Google Scholar] [CrossRef] [PubMed]
- Yeşilbağ, K.; Alpay, G.; Becher, P. Variability and Global Distribution of Subgenotypes of Bovine Viral Diarrhea Virus. Viruses 2017, 9, 128. [Google Scholar] [CrossRef] [PubMed]
- Chernick, A.; van der Meer, F. Evolution of Bovine viral diarrhea virus in Canada from 1997 to 2013. Virology 2017, 509, 232–238. [Google Scholar] [CrossRef] [PubMed]
Sample Name | BVDV Species/Subtype | |
---|---|---|
408TboUY/072014 | BVDV-1a | 1 |
409TboUY/072014 | BVDV-1a | 2 |
429TboUY/082014 | BVDV-1a | 3 |
430TboUY/082014 | BVDV-1a | 4 |
431TboUY/082014 | BVDV-1a | 5 |
432TboUY/082014 | BVDV-1a | 6 |
433FaUY/032014 | BVDV-1a | 7 |
434FaUY/032014 | BVDV-1a | 8 |
435FaUY/032014 | BVDV-1a | 9 |
437TboUY/042014 | BVDV-1a | 10 |
438TboUY/042014 | BVDV-1a | 11 |
653TboUY/082014 | BVDV-1a | 12 |
651TboUY/082014 | BVDV-1a | 13 |
652UYTbo/082014 | BVDV-1a | 14 |
588UYSa/2015 | BVDV-1a | 15 |
754UYAFA4/112015 | BVDV-1a | 16 |
1284UYTyT/022016 | BVDV-1a | 17 |
1532SJUY/042016 | BVDV-1a | 18 |
2144UY/2016 | BVDV-1a | 19 |
2145UY/2016 | BVDV-1a | 20 |
2146UY/2016 | BVDV-1a | 21 |
2147UY/2016 | BVDV-1a | 22 |
2148UY/2016 | BVDV-1a | 23 |
2402UYSJ/2016 | BVDV-1a | 24 |
2405UYSJ/2016 | BVDV-1a | 25 |
2514UYSJ/2016 | BVDV-1a | 26 |
3107UYCNIA/2016 | BVDV-1a | 27 |
3285RNUY/2017 | BVDV-1a | 28 |
3387UYCNES/2017 | BVDV-1a | 29 |
3397UYCNES/2017 | BVDV-1a | 30 |
3716UYCNIA/2017 | BVDV-1a | 31 |
3723UYCNIA/2017 | BVDV-1a | 32 |
3738UYLAV/2017 | BVDV-1a | 33 |
4552UYCNIA/2018 | BVDV-1a | 34 |
4678UYSOR/2018 | BVDV-1a | 35 |
4838UYCNES/2018 | BVDV-1a | 36 |
4852UYCNES/2018 | BVDV-1a | 37 |
4909UY/2018 | BVDV-1a | 38 |
5160 UYPAY/19 | BVDV-1a | 39 |
5258UY/2019 | BVDV-1a | 40 |
5354UYRN/2019 | BVDV-1a | 41 |
5356UYRN/2019 | BVDV-1a | 42 |
5419UYPAY/2019 | BVDV-1a | 43 |
5428UYPAY/2019 | BVDV-1a | 44 |
5436UY/2019 | BVDV-1a | 45 |
5578UY/2022 | BVDV-1a | 46 |
5582UYCNIA/2022 | BVDV-1a | 47 |
5584UY/2022 | BVDV-1a | 48 |
5615UY/2020 | BVDV-1a | 49 |
5651UY/2022 | BVDV-1a | 50 |
5684 UYTBO/2020 | BVDV-1a | 51 |
5685 UYTBO/2020 | BVDV-1a | 52 |
5767 UYSA/2023 | BVDV-1a | 53 |
5770 UYSA/2023 | BVDV-1a | 54 |
5772 UYSA/2023 | BVDV-1a | 55 |
5821 UY/2023 | BVDV-1a | 56 |
5822 UY/2023 | BVDV-1a | 57 |
5900 UY/2023 | BVDV-1a | 58 |
6067 UY/2024 | BVDV-1a | 59 |
6087 UY/2024 | BVDV-1a | 60 |
6092 TBO/2024 | BVDV-1a | 61 |
6094 UY/2024 | BVDV-1a | 62 |
6095 UY/2024 | BVDV-1a | 63 |
6096 UY/2024 | BVDV-1a | 64 |
6104 UY/2024 | BVDV-1a | 65 |
436FaUY/052014 | BVDV-1i | 66 |
5495UY/2019 | BVDV-1i | 67 |
5688UYTBO/2020 | BVDV-1e | 68 |
5691 UYTBO/2020 | BVDV-1e | 69 |
439RvUY/082014 | BVDV-2b | 70 |
2391UYRN/2016 | BVDV-2b | 71 |
2769UYRN/2016 | BVDV-2b | 72 |
3664UYCNIA/2017 | BVDV-2b | 73 |
3665UYCNIA/2017 | BVDV-2b | 74 |
4198UYCNES/2017 | BVDV-2b | 75 |
4511UYCNIA/2017 | BVDV-2b | 76 |
4516UYCNIA/2018 | BVDV-2b | 77 |
5280UY/2019 | BVDV-2b | 78 |
5281UY/2019 | BVDV-2b | 79 |
5282UY/2019 | BVDV-2b | 80 |
5290 UYPAY/19 | BVDV-2b | 81 |
5604UY/2020 | BVDV-2b | 82 |
5898 UY/2023 | BVDV-2b | 83 |
6097 UY/2024 | BVDV-2b | 84 |
5905 UY/2024 | not genotyped | 85 |
5690UYTBO/2020 | not genotyped | 86 |
5693 UYTBO/2020 | not genotyped | 87 |
Subtype | 5UTR/Npro | 5UTR | Total | ||
---|---|---|---|---|---|
BVDV-1a | 28 | 5 | 33 | ||
BVDV-1i | 1 | 0 | 1 | ||
BVDV-2b | 3 | 2 | 5 | ||
Maya et al., 2016, 2020 | Total | 32 | 7 | 39 | |
BVDV-1a | 25 | 7 | 32 | ||
BVDV-1e | 1 | 1 | 2 | ||
BVDV-1i | 0 | 1 | 1 | ||
BVDV-2b | 7 | 3 | 10 | ||
This study | Total | 33 | 12 | 45 | |
BVDV-1a | 53 | 12 | 65 | ||
BVDV-1e | 1 | 1 | 2 | ||
BVDV-1i | 1 | 1 | 2 | ||
BVDV-2b | 12 * | 3 * | 15 | ||
Maya et al., 2016, 2020 + this study | Total | 67 | 17 | 84 |
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. |
© 2025 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
Maya, L.; Castells, M.; Silveira, C.; Giannitti, F.; Merchioratto, I.; Barrandeguy, M.; Menchaca, A.; Colina, R. Epidemiology and Evolution of Bovine Viral Diarrhea Virus (BVDV) in Uruguay: A 10-Year Study. Viruses 2025, 17, 1374. https://doi.org/10.3390/v17101374
Maya L, Castells M, Silveira C, Giannitti F, Merchioratto I, Barrandeguy M, Menchaca A, Colina R. Epidemiology and Evolution of Bovine Viral Diarrhea Virus (BVDV) in Uruguay: A 10-Year Study. Viruses. 2025; 17(10):1374. https://doi.org/10.3390/v17101374
Chicago/Turabian StyleMaya, Leticia, Matias Castells, Caroline Silveira, Federico Giannitti, Ingryd Merchioratto, Maria Barrandeguy, Alejo Menchaca, and Rodney Colina. 2025. "Epidemiology and Evolution of Bovine Viral Diarrhea Virus (BVDV) in Uruguay: A 10-Year Study" Viruses 17, no. 10: 1374. https://doi.org/10.3390/v17101374
APA StyleMaya, L., Castells, M., Silveira, C., Giannitti, F., Merchioratto, I., Barrandeguy, M., Menchaca, A., & Colina, R. (2025). Epidemiology and Evolution of Bovine Viral Diarrhea Virus (BVDV) in Uruguay: A 10-Year Study. Viruses, 17(10), 1374. https://doi.org/10.3390/v17101374