Sequence analysis of the DA domain of glycoprotein E2 of pestiviruses isolated from beef cattle in Southern Brazil


The envelope glycoprotein E2 of pestiviruses is a major target for neutralizing antibodies. In this study, we analyzed the E2 DA domain of 43 pestiviruses from Southern Brazil. The isolates were identified as Bovine viral diarrhea virus (BVDV) subtypes 1a and 1b or BVDV-2b. Compared to reference strains, the BVDV-1 and -2 isolates had four and two mutations in the DA domain, respectively. All BVDV-2 isolates had a deletion of residues 724 and 725. All mutated amino acids in the BVDV isolates had the same aa substitution, and all were in previously identified antibody binding sites. It is possible that an immunity-mediated selection is acting on the pestiviruses circulating in Southern Brazil.

This is a preview of subscription content, access via your institution.

Fig. 1

Availability of data and material

The datasets generated and/or analyzed in the current study are available in the GenBank database and can also be made available by the corresponding author on reasonable request.


  1. 1.

    Lanyon SR, Hill FI, Reichel MP, Brownlie J (2014) Bovine viral diarrhoea: pathogenesis and diagnosis. Vet J 199: 201-209.

  2. 2.

    International Committee on Taxonomy of Viruses (2020) Taxonomy. . Accessed 05 June 2020

  3. 3.

    Yeşilbağ K, Alpay G, Becher P (2017) Variability and Global Distribution of Subgenotypes of Bovine Viral Diarrhea Virus. Viruses 9:128.

  4. 4.

    Schirrmeier H, Strebelow G, Depner K, Hoffmann B, Beer M (2004) Genetic and antigenic characterization of an atypical pestivirus isolate, a putative member of a novel pestivirus species. J Gen Virol 85: 3647-3652.

  5. 5.

    Stalder HP, Meier P, Pfaffen G, Wageck-Canal C, Rüfenacht J, Schaller P, Bachofen C, Marti S, Vogt HR, Peterhans E (2005) Genetic heterogeneity of pestiviruses of ruminants in Switzerland. Prev Vet Med 72: 37-41; discussion 215-219.

  6. 6.

    Decaro N, Lucente MS, Mari V, Cirone F, Cordioli P, Camero M, Sciarretta R, Losurdo M, Lorusso E, Buonavoglia C (2011) Atypical pestivirus and severe respiratory disease in calves, Europe. Emerg Infect Dis 17: 1549-1552.

  7. 7.

    Decaro N, Lucente MS, Mari V, Sciarretta R, Pinto P, Buonavoglia D, Martella V, Buonavoglia C (2012) Hobi-like pestivirus in aborted bovine fetuses. J Clin Microbiol 50: 509-512.

  8. 8.

    Ståhl K, Kampa J, Alenius S, Persson Wadman A, Baule C, Aiumlamai S, Belák S (2007) Natural infection of cattle with an atypical 'HoBi'-like pestivirus--implications for BVD control and for the safety of biological products. Vet Res 38: 517-523.

  9. 9.

    Dias RK, Cargnelutti JF, Weber MN, Canal CW, Bauermann FV, Ridpath JF, Weiblen R, Flores EF (2017) Antigenic diversity of Brazilian isolates of HoBi-like pestiviruses. Vet Microbiol 203: 221-228.

  10. 10.

    Monteiro FL, Cargnelutti JF, Braunig P, Folgueras-Flatschart AV, Santos NC, Pituco EM, Weiblen R, Flores EF (2018) Detection and genetic identification of pestiviruses in Brazilian lots of fetal bovine serum collected from 2006 to 2014. Pesq. Vet. Bras. 38(3):387-392.

  11. 11.

    Silveira S, Cibulski SP, Junqueira DM, Mósena ACS, Weber MN, Mayer FQ, Canal CW (2020) Phylogenetic and evolutionary analysis of HoBi-like pestivirus: Insights into origin and dispersal. Transbound Emerg Dis.

  12. 12.

    Tautz N, Tews BA, Meyers G (2015) The Molecular Biology of Pestiviruses. Adv Virus Res 93:47-160.

  13. 13.

    Ridpath JF, Bolin SR, Dubovi EJ (1994) Segregation of bovine viral diarrhea virus into genotypes. Virology 205: 66-74.

  14. 14.

    Becher P, Orlich M, Shannon AD, Horner G, König M, Thiel HJ (1997) Phylogenetic analysis of pestiviruses from domestic and wild ruminants. J Gen Virol 78 ( Pt 6): 1357-1366.

  15. 15.

    Vilcek S, Paton DJ, Durkovic B, Strojny L, Ibata G, Moussa A, Loitsch A, Rossmanith W, Vega S, Scicluna MT, Paifi V (2001) Bovine viral diarrhoea virus genotype 1 can be separated into at least eleven genetic groups. Arch Virol 146: 99-115.

  16. 16.

    Couvreur B, Letellier C, Collard A, Quenon P, Dehan P, Hamers C, Pastoret PP, Kerkhofs P (2002) Genetic and antigenic variability in bovine viral diarrhea virus (BVDV) isolates from Belgium. Virus Res 85: 17-28.

  17. 17.

    Pellerin, C, van den Hurk J, Lecomte J, Tijssen P (1994) Identification of a new group of bovine viral diarrhea virus strains associated with severe outbreaks and high mortalities. Virology 203: 260-268.

  18. 18.

    Deregt D, van Rijn PA, Wiens TY, van den Hurk J (1998) Monoclonal antibodies to the E2 protein of a new genotype (type 2) of bovine viral diarrhea virus define three antigenic domains involved in neutralization. Virus Res 57: 171-181.

  19. 19.

    Deregt D, Bolin SR, van den Hurk J, Ridpath JF, Gilbert SA (1998) Mapping of a type 1-specific and a type-common epitope on the E2 (gp53) protein of bovine viral diarrhea virus with neutralization escape mutants. Virus Res 53: 81-90.

  20. 20.

    Bauermann FV, Harmon A, Flores EF, Falkenberg SM, Reecy JM, Ridpath JF (2013) In vitro neutralization of HoBi-like viruses by antibodies in serum of cattle immunized with inactivated or modified live vaccines of bovine viral diarrhea viruses 1 and 2. Vet Microbiol 166: 242-245.

  21. 21.

    Bolin S, Moennig V, Kelso Gourley NE, Ridpath J (1988) Monoclonal antibodies with neutralizing activity segregate isolates of bovine viral diarrhea virus into groups. Brief report. Arch Virol 99:117–123.

    CAS  Article  PubMed  Google Scholar 

  22. 22.

    Donis RO, Corapi W, Dubovi EJ (1988) Neutralizing monoclonal antibodies to bovine viral diarrhoea virus bind to the 56K to 58K glycoprotein. J Gen Virol 69 ( Pt 1): 77-86.

  23. 23.

    Lindenbach BD, Murray CL, Thiel HJ, Rice CM (2013) Flaviviridae. In: Knipe DM, Howley PM (eds) Fields virology, 6th edn. Lippincott Williams & Wilkins, Philadelphia, pp 712–746

    Google Scholar 

  24. 24.

    El Omari K, Iourin O, Harlos K, Grimes JM, Stuart DI (2013) Structure of a pestivirus envelope glycoprotein E2 clarifies its role in cell entry. Cell Rep 3: 30-35.

  25. 25.

    Jelsma H, Loeffen WL, van Beuningen A, van Rijn PA (2013) Preliminary mapping of non-conserved epitopes on envelope glycoprotein E2 of Bovine viral diarrhea virus type 1 and 2. Vet Microbiol 166: 195-199.

  26. 26.

    Bolin SR (1993) Immunogens of bovine viral diarrhea virus. Vet. Microbiol. 37: 263-271.

  27. 27.

    Silveira S, Weber MN, Mósena AC, da Silva MS, Streck AF, Pescador CA, Flores EF, Weiblen R, Driemeier D, Ridpath JF, Canal CW (2017) Genetic diversity of Brazilian bovine pestiviruses detected between 1995 and 2014. Transbound Emerg Dis 64: 613-623.

  28. 28.

    Donoso A, Inostroza F, Celedón M, Pizarro-Lucero J (2018) Genetic diversity of bovine viral diarrhea virus from cattle in Chile between 2003 and 2007. BMC Vet Res 14: 314.

  29. 29.

    Jardim JC, Amaral BP, Martins M, Sebastian P, Heinemann MB, Cortez A, Weiblen R, Flores EF (2018) Respiratory signs, fever and lymphopenia in calves inoculated with Brazilian HoBi-like pestiviruses. Microb Pathog 123: 264-268.

  30. 30.

    Monteiro FL, Cargnelutti JF, Martins B, Noll JG, Weiblen R, Flores EF (2019) Detection of bovine pestiviruses in sera of beef calves by a RT-PCR based on a newly designed set of pan–bovine pestivirus primers. J Vet Diagn Invest 31(2):255-258.

  31. 31.

    Monteiro FL, Martins B, Cargnelutti JF, Noll JG, Weiblen R, Flores EF (2019) Genetic identification of pestiviruses from beef cattle in Southern Brazil. Braz J Microbiol 50(2):557-563.

  32. 32.

    Staden R (1996) The Staden sequence analysis package. Mol Biotechnol 5: 233-241.

  33. 33.

    Kumar S, Stecher G, Tamura K (2016) MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Mol Biol Evol 33:1870-1874.

  34. 34.

    Felsenstein J (1985) Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39: 783-791.

  35. 35.

    Hall TA (1999) BioEdit: a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Sympos Ser 41: 95-98.

  36. 36.

    Flores EF, Cargnelutti JF, Monteiro FL, Bauermann FV, Ridpath JF, Weiblen R (2018) A genetic profile of bovine pestiviruses circulating in Brazil (1998-2018). Anim Health Res Rev 19: 134-141.

  37. 37.

    Tijssen P, Pellerin C, Lecomte J, van den Hurk J (1996) Immunodominant E2 (gp53) sequences of highly virulent bovine viral diarrhea group II viruses indicate a close resemblance to a subgroup of border disease viruses. Virology 217: 356-361.

  38. 38.

    Abe Y, Tamura T, Torii S, Wakamori S, Nagai M, Mitsuhashi K, Mine J, Fujimoto Y, Nagashima N, Yoshino F, Sugita Y, Nomura T, Okamatsu M, Kida H, Sakoda Y (2016) Genetic and antigenic characterization of bovine viral diarrhea viruses isolated from cattle in Hokkaido, Japan. J Vet Med Sci 78: 61-70.

  39. 39.

    Li Y, Wang J, Kanai R, Modis Y (2013) Crystal structure of glycoprotein E2 from bovine viral diarrhea virus. PNAS 110:6805–6810.

    Article  PubMed  Google Scholar 

  40. 40.

    Tang F, Zhang C (2007) Evidence for positive selection on the E2 gene of bovine viral diarrhoea virus type 1. Virus Genes 35:629–634.

  41. 41.

    Mirosław P, Polak MP (2020) Variability of E2 protein-coding sequences of bovine viral diarrhea virus in Polish cattle. Virus Genes 56:515–521.

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. 42.

    Lang Y, Gao S, Du J, Shao J, Cong G, Lin T, Zhao F, Liu L, Chang H (2014) Polymorphic genetic characterization of E2 gene of bovine viral diarrhea virus in China. Vet Microbiol 174: 554-559.

  43. 43.

    Nagai M, Hayashi M, Sugita S, Sakoda Y, Mori M, Murakami T, Ozawa T, Yamada N, Akashi H (2004) Phylogenetic analysis of bovine viral diarrhea viruses using five different genetic regions. Virus Res 99:103–113.

Download references


Graduate students from the Programa de Pós-graduação em Medicina Veterinária, UFSM (COF, PSBO, JCGN). Carolina de Oliveira Freitas and Pablo Sebastian Britto de Oliveira thank the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Brazil) for their scholarships. Francielle Liz Monteiro thanks CNPq and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) (Brazil) for the scholarship. Jéssica Caroline Gomes Noll thanks CAPES for the scholarship. José Valter Joaquim Silva Júnior was supported by CAPES Postdoctoral Scholarship. Eduardo Furtado Flores (process 301414/2010-6) and Rudi Weiblen (process 304153/2014-1) were supported by CNPq research fellowships.


Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES). Finance code 001.

Author information




COF extracted the RNA, amplified the viral genes, analyzed gene sequenced, and assisted in the experimental design, interpretation of the results, and preparation of the manuscript; PBSO analyzed gene sequences, performed the analysis of amino acids and phylogeny, and assisted in the experimental design, interpretation of results, and preparation of the manuscript; FLM assisted in the amplification of viral genes, experimental design, and preparation of the manuscript; JCGN performed virus isolation and amplification; JVJr analyzed gene sequences, assisted in the analysis of phylogeny and amino acids, assisted in the experimental design, and interpretation of the results, and prepared the final version of the manuscript; RW assisted in the interpretation of results, experimental design and final review of the manuscript; EFF carried out the experimental design, assisted in the interpretation of the results, and preparation and final revision of the manuscript, and guided the study.

Corresponding author

Correspondence to Eduardo Furtado Flores.

Ethics declarations

Conflict of interest

The authors declare that they have no competing interests.

Ethical approval

Not applicable. This study did not involve animal experimentation. The samples used in the study came from the diagnostic service of Setor de Virologia, Universidade Federal de Santa Maria, Brazil.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Handling Editor: Patricia Aguilar.

Supplementary Information

Below is the link to the electronic supplementary material.

Sequences (and GenBank accession numbers) containing the DA domain of BVDV-1 (S1) analyzed in our study. (GB 4305 KB)

Sequences (and GenBank accession numbers) containing the DA domain of BVDV-2 (S2) analyzed in our study. (GB 2653 KB)

Sequences (and GenBank accession numbers) containing the DA domain of HoBiPeV (S3) analyzed in our study. (GB 555 KB)

Phylogeny based on partial 5'UTR sequences of pestivirus isolates. The analyzes were performed using MEGA 7 software and the neighbor-joining method. The evolutionary distances were calculated using the p-distance method. Bootstrap values were calculated using 1000 replicates. The BVDV-1 and BVDV-2 isolates sequenced in this study are indicated by symbols: open circles indicate the pestiviruses previously analyzed by Monteiro et al. [31], using primers HCV 90-368 or BVDV-2#3; filled circles indicate to the isolates from this study, amplified with primers BP189-389. (PDF 400 KB)

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

de Oliveira Freitas, C., de Oliveira, P.S.B., Monteiro, F.L. et al. Sequence analysis of the DA domain of glycoprotein E2 of pestiviruses isolated from beef cattle in Southern Brazil. Arch Virol 166, 1163–1170 (2021).

Download citation