Archives of Virology

, Volume 160, Issue 11, pp 2683–2691 | Cite as

Comparative analysis of the replication of bovine herpesvirus 1 (BHV1) and BHV5 in bovine-derived neuron-like cells

  • Tereza C. Cardoso
  • Helena. L. Ferreira
  • Lucas H. Okamura
  • Bruna R. S. M. Oliveira
  • Ana Carolina G. Rosa
  • Roberto Gameiro
  • Eduardo F. Flores
Original Article


Members of the subfamily Alphaherpesvirinae use the epithelium of the upper respiratory and/or genital tract as preferential sites for primary replication. However, bovine herpesvirus 5 (BoHV5) is neurotropic and neuroinvasive and responsible for meningoencephalitis in cattle and in animal models. A related virus, BoHV1 has also been occasionally implicated in natural cases of neurological infection and disease in cattle. The aim of the present study was to assess the in vitro effects of BoHV1 and BoHV5 replication in neuron-like cells. Overall, cytopathic effects, consisting of floating rounded cells, giant cells and monolayer lysis, induced by both viruses at 48 h postinfection (p.i.) resulted in a loss of cell viability and high virus titres (r = 0.978). The BoHV1 Cooper strain produced the lowest titres in neuron-like cells, although viral DNA was detected in infected cells during all experiments. Virus replication in infected cells was demonstrated by immunocytochemistry, flow cytometry and qPCR assays. BoHV antigens were better visualized at 48 h p.i. and flow cytometry analysis showed that SV56/90 and Los Angeles antigens were present at higher levels. In spite of the fact that BoHV titres dropped at 48 h p.i, viral DNA remained detectable until 120 h p.i. Sensitive TUNEL (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling) and annexin V assays were used to identify apoptosis. BoHV5 induced death in approximately 50 % of cells within 24 h p.i., similar to what has been observed for BoHV1 Los Angeles. Infection with the BoHV1 Cooper strain resulted in 26.37 % of cells being in the early stages of apoptosis; 63.69 % of infected cells were considered viable. Modulation of mitochondrial function, as measured by mitochondrial membrane depolarization, was synchronous with the virus replication cycle, cell viability and virus titres at 48 h p.i. Our results indicate that apoptosis plays an important role in preventing neuronal death and provides a bovine-derived in vitro system to study herpesvirus-neuron interactions.


Mitochondrial Membrane Potential Trigeminal Ganglion Cytopathic Effect Uninfected Cell Trigeminal Ganglion Neuron 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.



The authors thank the Fundação Amparo à Pesquisa do Estado de São Paulo (Grants 2012/16715-6) for their support. EF Flores and TC Cardoso are recipients of CNPq (Brazilian Council for Research) funding.


  1. 1.
    Brenner MPC, Silva-Frade C, Ferrarezi MC, Garcia AF, Flores EF, Cardoso TC (2012) Evaluation of developmental changes in bovine in vitro produced embryos following exposure to bovine herpesvirus 5. Rep Biol Endrocrinol 10:53CrossRefGoogle Scholar
  2. 2.
    Cardoso TC, Novais JB, Antello TF, Silva-Frade C, Ferrarezi MC, Ferrari HF, Gameiro R, Flores EF (2012) Susceptibility of neuron-like cells derived from bovine Wharton’s jelly to bovine herpesvirus type 5 infections. BMC Vet Res 8:242PubMedCentralCrossRefPubMedGoogle Scholar
  3. 3.
    Christensen J, Steain M, Slobedman B, Abendroth A (2011) Differentiated neuroblastoma cells provide a highly efficient model for studies of productive varicella-Zoster virus infection of neuronal cells. J Virol 85:8436–8442PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Cymerys J, Slonska A, Godlewski MM, Golke A, Tucholska A, Chmielewska A, Banbura MW (2012) Apoptotic and necrotic changes in cultured murine neurons infected with equid herpesvirus 1. Acta Virol 56:39–48CrossRefPubMedGoogle Scholar
  5. 5.
    Davison AJ, Eberle R, Ehlers B, Hayward GS, McGeoch DJ, Minson AC, Pellet PE, Roizman B, Studdert MJ, Thiry E (2009) The order Herpesvirales. Arch Virol 154:171–177PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Davison AJ (2010) Herpesvirus systematics. Vet Microbiol 143:52–69PubMedCentralCrossRefPubMedGoogle Scholar
  7. 7.
    Del Médico Zajac MP, Ladelfa MF, Kotsias F, Muylkens B, Thiry J, Thiry E, Romera SA (2010) Biology of bovine herpesvirus 5. Vet J 184:138–145CrossRefPubMedGoogle Scholar
  8. 8.
    Delhon GA, González MJ, Murcia PR (2002) Susceptibility of sensory neurons to apoptosis following infection by bovine herpesvirus type 1. J Gen Virol 83:2257–2267CrossRefPubMedGoogle Scholar
  9. 9.
    Diallo IS, Corney BG, Rodwell BJ (2011) Detection and differentiation of bovine herpesvirus 1 and 5 using multiplex real-time polymerase chain reaction. J Virol Meth 175:46–52CrossRefGoogle Scholar
  10. 10.
    Egan KP, Wu S, Wigdahl Jennings SR (2013) Immunological control of herpes simplex virus infections. J Neurovirol 19:328–345PubMedCentralCrossRefPubMedGoogle Scholar
  11. 11.
    Ferrari HF, Luvizotto MCR, Rahal P, Cardoso TC (2007) Detection of bovine herpesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue based diagnostic test of bovine encephalitis. J Virol Meth 143:335–340CrossRefGoogle Scholar
  12. 12.
    Griffin BD, Verweij MC, Wiertz EJHJ (2010) Herpesvirus and immunity: art of invasion. Vet Microbiol 143:89–100CrossRefPubMedGoogle Scholar
  13. 13.
    Hay S, Kannourakis G (2002) A time to kill: viral manipulation of the cell death program. J Gen Virol 83:1547–1564CrossRefPubMedGoogle Scholar
  14. 14.
    Hood C, Cunningham AI, Sloberdman B, Boadle RA, Abendroth A (2003) Varicella-zooster virus–infected human sensory neurons are resistant to apoptosis, yet human foreskin fibroblasts are susceptible: evidence for cell-type-specific apoptotic response. J Virol 77:12852–12864PubMedCentralCrossRefPubMedGoogle Scholar
  15. 15.
    Scott Iain (2010) The role of mitochondria in the mammalian antiviral defense system. Mitochondrion 10:316–320PubMedCentralCrossRefPubMedGoogle Scholar
  16. 16.
    Jones C, Silva LF, Sinani D (2011) Regulation of the latency-reactivation cycle by products encoded by bovine herpesvirus 1 (BHV-1) latency-related gene. J Neurol 17:535–545Google Scholar
  17. 17.
    Lovato L, Inman M, Henderson G, Doster A, Jones C (2003) Infection pf cattle with a bovine herpesvirus 1 strain that contains a mutation in latency-related gene leads to increased apoptosis in trigeminal ganglia during the transition from acute infection to latency. J Virol 77:4848–4857PubMedCentralCrossRefPubMedGoogle Scholar
  18. 18.
    Machado GF, Bernardi F, Hosomi FYM, Peiró JR, Weiblen R, Roehe PM, Alessi AC, Melo GD, Ramos AT, Maiorka PC (2013) Bovine herpesvirus-5 infection in a rabbit experimental model: immunohistochemical study of cellular response in the CNS. Microb Pathog 57:10–16CrossRefPubMedGoogle Scholar
  19. 19.
    Marin MS, Leunda MR, Verna AE, Faverin C, Pérez SE, Odeón AC (2012) In vitro replication of bovine herpesvirus types 1 and 5. J Virol Meth 181:80–85CrossRefGoogle Scholar
  20. 20.
    Montagnaro S, Ciarcia R, De Martinis C, Pacilio C, Sasso S, Puzio MV, De Angelis M, Pagnini U, Boffo S, Kenez I, Iovanne G, Giordano A (2013) Modulation of apoptosis by caprine herpesvirus 1 infection in a neuronal cell line. J Cell Biochem 114:2809–2822CrossRefPubMedGoogle Scholar
  21. 21.
    Ohta A, Nishiyama Y (2011) Mitochondria and viruses. Mitochondrion 11:1–12CrossRefPubMedGoogle Scholar
  22. 22.
    Paroli M, Schiafella E, Di Rosa F, Barnaba V (2000) Persisting viruses and autoimmunity. J Neuroimmunol 107:201–204CrossRefPubMedGoogle Scholar
  23. 23.
    Pugazhenthi S, Nair S, Velmurugan K, Liang Q, Mahalingam R, Cohrs RJ, Nagel MA, Gilden D (2011) Varicella-Zoster virus infection of differentiated human neural stem cells. J Virol 85:6678–6686PubMedCentralCrossRefPubMedGoogle Scholar
  24. 24.
    Silva MS, Brum MCS, Loreto ELS, Weiblen R, Flores EF (2007) Molecular and antigenic characterization of Brazilian bovine herpesvirus type 1 isolates recovered from the brain of cattle with neurological disease. Virus Res 129:191–199CrossRefPubMedGoogle Scholar
  25. 25.
    Silva-Frade C, Gameiro R, Martins A Jr, Cardoso TC (2010) Apoptotic and developmental effects of bovine herpesvirus type-5 infection on in vitro-produced bovine embryos. Theriogenology 74:1296–1303CrossRefPubMedGoogle Scholar
  26. 26.
    Silva-Frade C, Gameiro R, Okamura LH, Flores EF, Cardoso TC (2014) Programmed cell death-associated gene transcripts in bovine embryos exposed to bovine herpesvirus type 5. Mol Cell Probes 28:113–117CrossRefPubMedGoogle Scholar
  27. 27.
    Steukers L, Vandekerckhove AP, den Broeck WV, Glorieux S, Nauwynck HJ (2011) Comparative analysis of replication characteristics of BoHV-1 subtypes in bovine respiratory and genital mucosa explants: phylogenetic enlightenment. Vet Res 42:33PubMedCentralCrossRefPubMedGoogle Scholar
  28. 28.
    Varela APM, Holz CL, Cibulski SP, Teixeira TF, Antunes DA, Franco AC, Roehe LR, Oliveira MT, Campos FS, Dezen D, Cenci D, Brito WD, Rohe PM (2010) Neutralizing antibodies to bovine herpesvirus types 1 (BoHV-1) and 5 (BoHV-5) and its subtypes. Vet Microbiol 142:254–260CrossRefPubMedGoogle Scholar
  29. 29.
    West AP, Shadel GS, Ghosh S (2011) Mitochondria in innate immune responses. Nat Rev 11:389–402CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Wien 2015

Authors and Affiliations

  • Tereza C. Cardoso
    • 1
  • Helena. L. Ferreira
    • 3
  • Lucas H. Okamura
    • 1
  • Bruna R. S. M. Oliveira
    • 1
  • Ana Carolina G. Rosa
    • 1
  • Roberto Gameiro
    • 2
  • Eduardo F. Flores
    • 4
  1. 1.Laboratory of Animal Virology and Cell CultureUniversidade Estadual PaulistaAraçatubaBrazil
  2. 2.Embryology of Domestic Animals, College of Veterinary MedicineUniversidade Estadual PaulistaAraçatubaBrazil
  3. 3.Departamento de Medicina VeterináriaFZEA-USPPirassunungaBrazil
  4. 4.Virology SectionFederal University of Santa MariaSanta MariaBrazil

Personalised recommendations