Proposal of Model for Evaluation of Viral Kinetics of African/Asian/Brazilian—Zika virus Strains (Step Growth Curve) in Trophoblastic Cell Lines
<p>Experiment with viral kinetics. Steps 1 and 2 represent cell cultures in plates of six-wells plates where they were inoculated with ZIKV and collated after each hour post infection (hpi), and the monolayer and supernatant underwent qRT-PCR analysis. In step 3, the viral growth curve was elaborated by Ellis and Delbruck [<a href="#B20-viruses-15-01446" class="html-bibr">20</a>], whereas Burleson [<a href="#B23-viruses-15-01446" class="html-bibr">23</a>] presented the graphical description of the phases of the replicative cycle related to enveloped viruses, considering the time versus unit of infectious particles formed. Based on the models by Delbruck and Ellis, and Burleson, the curve can be divided into two parts. The first is characterized by the (I) adsorption, and (II) penetration and disassembly of viral particles: onset period of transcription, translation, and replication of the viral genome. In this stage, few viruses are detectable, and the most accurate verification of their presence is by means of qRT-PCR or immunofluorescence. The second part involves the assembly, (III) maturation and (IV) release of the viral progeny, in addition to its detection by different molecular and cellular methods. Both phases occur concurrently.</p> "> Figure 2
<p>Image obtained by inverted-light optical microscope with magnification at 200 μm (20×). The data obtained in our study should be analyzed independently for each ZIKV strain as we used different MOIs due to the lack of ZIKV-IEC titers to reach MOI = 1. The monolayer of infected cells (red arrow), from the viral kinetics experiment, of (<b>a</b>) ZIKV-MR766 low passage (African) and (<b>b</b>) ZIKV-IEC-Paraíba (Asian-Brazilian) strains hpi 96 h post infection. Comparative observation between mock and infected cells showed the characteristic cytopathic effects (CPEs) of ZIKV. Furthermore, such features seen, under the optical microscope, in the BeWo, BeWo + fork, and HTR-8 were the same, as noticed in the HuH-7 control lineage: monolayer detachment; focal degeneration with rounded and refractory cells; partial and total destruction of monolayer inoculated; generation of cellular debris; morphological alterations, edema, and crowding of cells. The non-infected BeWo lineage, as observed in its the daily maintenance of it, began to detach from the monolayer after three days. Such cells had rapid cell division and expansion. When they reached 100% of space occupation, the oldest spontaneously separated from the monolayer.</p> "> Figure 3
<p>Rate graphic of PFU/mL relation between extra- and intracellular viral kinetics of BeWo, BeWo treated with forskolin, HTR-8, HuH-7 infected by ZIKV-MR766 low passage, and ZIKV-IEC-Paraíba (<a href="#app8-viruses-15-01446" class="html-app">Appendix G</a>). The abscissa shows the infected lineages during the period of post infection (hpi) from 2 to 144, while the ordinate indicates a relation between extra- and intracellular Log<sub>10</sub>PFU/mL. The data obtained in our study should be analyzed independently for each ZIKV strain as we used different MOIs due to the lack of ZIKV-IEC titers to reach MOI = 1.</p> "> Figure A1
<p>Experiment with standard curve. (1) The standard curve established a direct relation between C<sub>T</sub> and PFU/mL, obtained from the viral stock titration of ZIKV-MR766 and ZIKV-IEC-Paraiba. Such titration was performed in triplicate and its mean values were gotten. The serial dilution for the titration started from 10<sup>−1</sup> up to 10<sup>−11</sup> fraction. (2) In a 24-well plate and in duplicate for each fraction. However, we collected the total volume for qRT-PCR because all cells generally die in the first fractions. (3) The extraction followed the TRIzol<sup>®</sup>Reagent (Invitrogen™ Cat#15596026) protocol. Extracted samples were quantified in the NanoDrop™ spectrophotometer and normalized to an average value of 200 ng/μL RNA contraction. The reaction and primers for qRT-PCR followed the protocol published by Lanciotti et al., 2008 [<a href="#B31-viruses-15-01446" class="html-bibr">31</a>]. (4) After obtaining the C<sub>T</sub> values for each fraction, we calculated the corresponding values of PFU/mL with them.</p> "> Figure A2
<p>Graphic Results of Standard Curves by qRT-PCR.</p> "> Figure A3
<p>Graphic Results of Complete Viral Kinetics.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Viral Isolation and Formation of Viral Stocks
2.2. Cells Cultures
2.3. Standard Curve for PFU/mL Determination
2.4. Viral Kinetics (Step Growth Curve)
3. Results
3.1. Inoculation of ZIKV-MR766lp (a)
3.2. Inoculation of ZIKV-IEC-Paraíba (b)
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Appendix A. BeWo Treated with Forskolin
Appendix B. Adaptation of qRT-PCR Performed by Lanciotti et al. 2008 [31]
Appendix C. Viral Kinetics
Appendix D. Standard Curve|Direct Relation between CT and PFU/mL
Appendix E. Standard Curve|Calculation of Direct Relation between CT and PFU/mL
Appendix F. Conversion of CT Values into PFU/mL of the Viral Kinetics Graph
Strain/Replicate (PFU/mL) | First | Second | Third | Average |
ZIKV-MR766 low passage | 1.57 × 108 | 2.05 × 108 | 1.43 × 108 | 1.68 × 108 |
ZIKV-IEC-Paraíba | 1.30 × 106 | 1.40 × 106 | 1.68 × 106 | 1.46 × 108 |
Appendix G. Calculation of the Extra- and Intracellular Ratio
Appendix H. Figure of Standard Curves
Appendix I. Figure of Complete Viral Kinetics
References
- Dick, G.W.; Kitchen, S.F.; Haddow, A.J. Zika virus (I). Isolations and serological specificity. Trans. R. Soc. Trop. Med. Hyg. 1952, 46, 509–520. [Google Scholar] [CrossRef]
- Dick, G.W.; Kitchen, S.F.; Haddow, A.J. Zika virus (II). Pathogenicity and physical properties. Trans. R. Soc. Trop. Med. Hyg. 1952, 46, 521–534. [Google Scholar] [CrossRef]
- Dick, G.W. Epidemiological notes on some viruses isolated in Uganda (Yellow fever, Rift Valley fever, Bwamba fever, West Nile, Mengo, Semliki Forest, Bunyamwera, Ntaya, Uganda S and Zika viruses). Trans. R. Soc. Trop. Med. Hyg. 1953, 47, 13–48. [Google Scholar] [CrossRef] [Green Version]
- Simpson, D.I. Zika virus infection in man. Trans. R. Soc. Trop. Med. Hyg. 1964, 58, 335–338. [Google Scholar] [CrossRef] [PubMed]
- Marchette, N.J.; Garcia, R.; Rudnick, A. Isolation of Zika virus from Aedes aegypti mosquitoes in Malaysia. Am. J. Trop. Med. Hyg. 1969, 18, 411–415. [Google Scholar] [CrossRef]
- Olson, J.G.; Ksiazek, T.G. Zika virus, a cause of fever in Central Java, Indonesia. Trans. R. Soc. Trop. Med. Hyg. 1981, 75, 389–393. [Google Scholar] [CrossRef] [PubMed]
- Posen, H.J.; Keystone, J.S.; Gubbay, J.B.; Morris, S.K. Epidemiology of Zika virus, 1947–2007. BMJ Glob. Health 2016, 1, e000087. [Google Scholar] [CrossRef] [Green Version]
- Musso, D.; Gubler, D.J. Zika virus. Clin. Microbiol. Rev. 2016, 29, 487–524. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Masmejan, S.; Musso, D.; Vouga, M.; Pomar, L.; Dashraath, P.; Stojanov, M.; Panchaud, A.; Baud, D. Zika virus. Pathogens 2020, 9, 898. [Google Scholar] [CrossRef] [PubMed]
- Musso, D.; Ko, A.I.; Baud, D. Zika virus infection—After the pandemic. N. Engl. J. Med. 2019, 381, 1444–1457. [Google Scholar] [CrossRef] [PubMed]
- Pardy, R.D.; Richer, M.J. Zika virus Pathogenesis: From Early Case Reports to Epidemics. Viruses 2019, 11, 886. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Martines, R.B.; Bhatnagar, J.; de Oliveira Ramos, A.M.; Davi, H.P.; Iglezias, S.D.; Kanamura, C.T.; Keating, M.K.; Hale, G.; Silva-Flannery, L.; Muehlenbachs, A.; et al. Pathology of congenital Zika syndrome in Brazil: A case series. Lancet 2016, 388, 898–904. [Google Scholar] [CrossRef] [Green Version]
- Miner, J.J.; Diamond, M.S. Zika virus pathogenesis and tissue tropism. Cell Host Microbe 2017, 21, 134–142. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Rabelo, K.; De Souza, L.J.; Salomão, N.G.; Machado, L.N.; Pereira, P.G.; Portari, E.A.; Basílio-de-Oliveira, R.; Dos Santos, F.B.; Neves, L.D.; Morgade, L.F.; et al. Zika induces human placental damage and inflammation. Front. Immunol. 2020, 1, 2146. [Google Scholar] [CrossRef]
- Hasan, S.S.; Sevvana, M.; Kuhn, R.J.; Rossmann, M.G. Structural biology of Zika virus and other flaviviruses. Nat. Struct. Mol. Biol. 2018, 25, 13–20. [Google Scholar] [CrossRef]
- Xu, Y.; He, Y.; Momben-Abolfath, S.; Vertrees, D.; Li, X.; Norton, M.G.; Struble, E.B. Zika virus Infection and Antibody Neutralization in FcRn Expressing Placenta and Engineered Cell Lines. Vaccines 2022, 10, 2059. [Google Scholar] [CrossRef]
- Cao, B.; Diamond, M.S.; Mysorekar, I.U. Maternal-fetal transmission of Zika virus: Routes and signals for infection. J. Interferon Cytokine Res. 2017, 37, 287–294. [Google Scholar] [CrossRef] [Green Version]
- Mysorekar, I.U.; Diamond, M.S. Modeling Zika virus infection in pregnancy. N. Engl. J. Med. 2016, 375, 481–484. [Google Scholar] [CrossRef]
- Carrera, J.; Trenerry, A.M.; Simmons, C.P.; Mackenzie, J.M. Flavivirus replication kinetics in early-term placental cell lines with different differentiation pathways. Virol. J. 2021, 18, 1–9. [Google Scholar] [CrossRef]
- Ellis, E.L.; Delbruck, M. The growth of bacteriophage. J. Gen. Physiol. 1939, 22, 365–384. [Google Scholar] [CrossRef] [Green Version]
- Keogh, B.P. Adsorption, latent period and burst size of phages of some strains of lactic streptococci. J. Dairy Res. 1973, 40, 303–309. [Google Scholar] [CrossRef]
- Falke, D. Virologia; E.P.U. Springer EDUSP: São Paulo, Brasil, 1979; pp. 13–34. [Google Scholar]
- Burleson, F.G.; Chamber, T.M.; Widebrauk, D. Virology: A Laboratory Manual; Academic Press, Inc.: San Diego, CA, USA, 1992; pp. 100–106. [Google Scholar]
- Mautner, L.; Hoyos, M.; Dangel, A.; Berger, C.; Ehrhardt, A.; Baiker, A. Replication kinetics and infectivity of SARS-CoV-2 variants of concern in common cell culture models. Virol. J. 2022, 19, 1–11. [Google Scholar] [CrossRef] [PubMed]
- Kuno, G.; Chang, G.J. Full-length sequencing and genomic characterization of Bagaza, Kedougou, and Zika viruses. Arch. Virol. 2007, 152, 687–696. [Google Scholar] [CrossRef] [PubMed]
- Haddow, A.D.; Schuh, A.J.; Yasuda, C.Y.; Kasper, M.R.; Heang, V.; Huy, R.; Guzman, H.; Tesh, R.B.; Weaver, S.C. Genetic characterization of Zika virus strains: Geographic expansion of the Asian lineage. PLoS Negl. Trop. Dis. 2012, 6, e1477. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Faye, O.; Freire, C.C.; Iamarino, A.; Faye, O.; de Oliveira, J.V.; Diallo, M.; Zanotto, P.M.; Sall, A.A. Molecular evolution of Zika virus during its emergence in the 20th century. PLoS Negl. Trop. Dis. 2014, 8, e2636. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Faria, N.R.; Azevedo, R.D.; Kraemer, M.U.; Souza, R.; Cunha, M.S.; Hill, S.C.; Thézé, J.; Bonsall, M.B.; Bowden, T.A.; Rissanen, I.; et al. Zika virus in the Americas: Early epidemiological and genetic findings. Science 2016, 352, 345–349. [Google Scholar] [CrossRef] [Green Version]
- Aubry, F.; Jacobs, S.; Darmuzey, M.; Lequime, S.; Delang, L.; Fontaine, A.; Jupatanakul, N.; Miot, E.F.; Dabo, S.; Manet, C.; et al. Recent African strains of Zika virus display higher transmissibility and fetal pathogenicity than Asian strains. Nat. Commun. 2021, 12, 916. [Google Scholar] [CrossRef]
- Timenetsky, J.; Santos, L.M.; Buzinhani, M.; Mettifogo, E. Detection of multiple mycoplasma infection in cell cultures by PCR. Braz. J. Med. Biol. Res. 2006, 39, 907–914. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Lanciotti, R.S.; Kosoy, O.L.; Laven, J.J.; Velez, J.O.; Lambert, A.J.; Johnson, A.J.; Stanfield, S.M.; Duffy, M.R. Genetic and serologic properties of Zika virus associated with an epidemic, Yap State, Micronesia, 2007. Emerg. Infect. Dis. 2008, 14, 1232–1239. [Google Scholar] [CrossRef]
- Orendi, K.; Gauster, M.; Moser, G.; Meiri, H.; Huppertz, B. The choriocarcinoma cell line BeWo: Syncytial fusion and expression of syncytium-specific proteins. Reproduction 2010, 40, 759–766. [Google Scholar] [CrossRef] [Green Version]
- Sheridan, M.A.; Yunusov, D.; Balaraman, V.; Alexenko, A.P.; Yabe, S.; Verjovski-Almeida, S.; Schust, D.J.; Franz, A.W.; Sadovsky, Y.; Ezashi, T.; et al. Vulnerability of primitive human placental trophoblast to Zika virus. Proc. Natl. Acad. Sci. USA 2017, 114, 1587–1596. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tabata, T.; Petitt, M.; Puerta-Guardo, H.; Michlmayr, D.; Wang, C.; Fang-Hoover, J.; Harris, E.; Pereira, L. Zika virus targets different primary human placental cells, suggesting two routes for vertical transmission. Cell Host Microbe 2016, 20, 155–166. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Tabata, T.; Petitt, M.; Puerta-Guardo, H.; Michlmayr, D.; Harris, E.; Pereira, L. Zika virus replicates in proliferating cells in explants from first-trimester human placentas, potential sites for dissemination of infection. J. Infect. Dis. 2018, 217, 1202–1213. [Google Scholar] [CrossRef] [Green Version]
- Gilbert, S.F.; Barresi, M.J.F. Developmental Biology, 11th ed.; Sinauer Associates, Inc.: Sunderland, MA, USA, 2016; pp. 143–180. [Google Scholar]
- Arora, N.; Sadovsky, Y.; Dermody, T.S.; Coyne, C.B. Microbial vertical transmission during human pregnancy. Cell Host Microbe 2017, 21, 561–567. [Google Scholar] [CrossRef] [PubMed]
- Sheridan, M.A.; Balaraman, V.; Schust, D.J.; Ezashi, T.; Roberts, R.M.; Franz, A.W. African and Asian strains of Zika virus differ in their ability to infect and lyse primitive human placental trophoblast. PLoS ONE 2018, 13, e0200086. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- León-Juárez, M.; Martínez-Castillo, M.; González-García, L.D.; Helguera-Repetto, A.C.; Zaga-Clavellina, V.; García-Cordero, J.; Flores-Pliego, A.; Herrera-Salazar, A.; Vázquez-Martínez, E.R.; Reyes-Muñoz, E. Cellular and molecular mechanisms of viral infection in the human placenta. Pathog. Dis. 2017, 75, 1–15. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- El Costa, H.; Gouilly, J.; Mansuy, J.M.; Chen, Q.; Levy, C.; Cartron, G.; Veas, F.; Al-Daccak, R.; Izopet, J.; Jabrane-Ferrat, N. ZIKA virus reveals broad tissue and cell tropism during the first trimester of pregnancy. Sci. Rep. 2016, 6, 1–9. [Google Scholar] [CrossRef] [Green Version]
- Simister, N.E. Placental transport of immunoglobulin G. Vaccine 2003, 21, 3365–3369. [Google Scholar] [CrossRef]
- Quicke, K.M.; Bowen, J.R.; Johnson, E.L.; McDonald, C.E.; Ma, H.; O’Neal, J.T.; Rajakumar, A.; Wrammert, J.; Rimawi, B.H.; Pulendran, B.; et al. Zika virus infects human placental macrophages. Cell Host Microbe 2016, 20, 83–90. [Google Scholar] [CrossRef] [Green Version]
- Liu, Z.; Zhang, Y.; Cheng, M.; Ge, N.; Shu, J.; Xu, Z.; Su, X.; Kou, Z.; Tong, Y.; Qin, C.; et al. A single nonsynonymous mutation on ZIKV E protein-coding sequences leads to markedly increased neurovirulence in vivo. Virol. Sin. 2022, 37, 115–126. [Google Scholar] [CrossRef]
- Yuan, L.; Huang, X.Y.; Liu, Z.Y.; Zhang, F.; Zhu, X.L.; Yu, J.Y.; Ji, X.; Xu, Y.P.; Li, G.; Li, C.; et al. A single mutation in the prM protein of Zika virus contributes to fetal microcephaly. Science 2017, 358, 933–936. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- King, E.L.; Irigoyen, N. Zika virus and Neuropathogenesis: The Unanswered Question of Which Strain Is More Prone to Causing Microcephaly and Other Neurological Defects. Front. Cell. Neurosci. 2021, 15, 1–14. [Google Scholar] [CrossRef] [PubMed]
- Cugola, F.R.; Fernandes, I.R.; Russo, F.B.; Freitas, B.C.; Dias, J.L.; Guimarães, K.P.; Benazzato, C.; Almeida, N.; Pignatari, G.C.; Romero, S.; et al. The Brazilian Zika virus strain causes birth defects in experimental models. Nature 2016, 534, 267–271. [Google Scholar] [CrossRef] [Green Version]
- Adibi, J.J.; Marques, E.T., Jr.; Cartus, A.; Beigi, R.H. Teratogenic effects of the Zika virus and the role of the placenta. Lancet 2016, 387, 1587–1590. [Google Scholar] [CrossRef] [Green Version]
- Meaney-Delman, D.; Oduyebo, T.; Polen, K.N.; White, J.L.; Bingham, A.M.; Slavinski, S.A.; Heberlein-Larson, L.; St George, K.; Rakeman, J.L.; Hills, S.; et al. Prolonged detection of Zika virus RNA in pregnant women. Obstet. Gynecol. 2016, 128, 724–730. [Google Scholar] [CrossRef] [PubMed] [Green Version]
- Suy, A.; Sulleiro, E.; Rodó, C.; Vázquez, É.; Bocanegra, C.; Molina, I.; Esperalba, J.; Sánchez-Seco, M.P.; Boix, H.; Pumarola, T.; et al. Prolonged Zika virus viremia during pregnancy. N. Engl. J. Med. 2016, 375, 2611–2613. [Google Scholar] [CrossRef]
- Muthuraj, P.G.; Sahoo, P.K.; Kraus, M.; Bruett, T.; Annamalai, A.S.; Pattnaik, A.; Pattnaik, A.K.; Byrareddy, S.N.; Natarajan, S.K. Zika virus infection induces endoplasmic reticulum stress and apoptosis in placental trophoblasts. Cell Death Discov. 2021, 7, 24. [Google Scholar] [CrossRef]
- Da Silva, S.; Martins, D.O.S.; Jardim, A.C.G. A review of the ongoing research on Zika virus treatment. Viruses 2018, 10, 255. [Google Scholar] [CrossRef] [Green Version]
- Saiz, J.C.; Oya, N.J.D.; Blázquez, A.B.; Escribano-Romero, E.; Martín-Acebes, M.A. Host-directed antivirals: A realistic alternative to fight Zika virus. Viruses 2018, 10, 453. [Google Scholar] [CrossRef] [Green Version]
- Giraldo, M.I.; Gonzalez-Orozco, M.; Rajsbaum, R. Pathogenesis of Zika virus Infection. Annu. Rev. Pathol. Mech. Dis. 2023, 18, 181–203. [Google Scholar] [CrossRef]
- Pena, L.J.; Guarines, K.M.; Silva, A.J.D.; Leal, L.R.S.; Félix, D.M.; Silva, A.; de Oliveira, S.A.; Ayres, C.F.J.; Júnior, A.S.; de Freitas, A.C. In vitro and in vivo models for studying Zika virus biology. J. Gen. Virol. 2018, 99, 1529–1550. [Google Scholar] [CrossRef] [PubMed]
- Msheik, H.; Azar, J.; El Sabeh, M.; Abou-Kheir, W.; Daoud, G. HTR-8/SVneo: A model for epithelial to mesenchymal transition in the human placenta. Placenta 2020, 90, 90–97. [Google Scholar] [CrossRef] [PubMed]
- Liu, F.; Soares, M.J.; Audus, K.L. Permeability properties of monolayers of the human trophoblast cell line BeWo. Am. J. Physiol. Cell Physiol. 1997, 273, C1596–C1604. [Google Scholar] [CrossRef] [PubMed]
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Barbosa, M.D.; Costa, A.; Prieto-Oliveira, P.; Andreata-Santos, R.; Peter, C.M.; Zanotto, P.M.A.; Janini, L.M.R. Proposal of Model for Evaluation of Viral Kinetics of African/Asian/Brazilian—Zika virus Strains (Step Growth Curve) in Trophoblastic Cell Lines. Viruses 2023, 15, 1446. https://doi.org/10.3390/v15071446
Barbosa MD, Costa A, Prieto-Oliveira P, Andreata-Santos R, Peter CM, Zanotto PMA, Janini LMR. Proposal of Model for Evaluation of Viral Kinetics of African/Asian/Brazilian—Zika virus Strains (Step Growth Curve) in Trophoblastic Cell Lines. Viruses. 2023; 15(7):1446. https://doi.org/10.3390/v15071446
Chicago/Turabian StyleBarbosa, Márcia Duarte, Anderson Costa, Paula Prieto-Oliveira, Robert Andreata-Santos, Cristina M. Peter, Paolo M. A. Zanotto, and Luiz Mario Ramos Janini. 2023. "Proposal of Model for Evaluation of Viral Kinetics of African/Asian/Brazilian—Zika virus Strains (Step Growth Curve) in Trophoblastic Cell Lines" Viruses 15, no. 7: 1446. https://doi.org/10.3390/v15071446