Abstract
Research of cyprinid herpesvirus 3 (CyHV-3) is focused on the infection mechanism and disease development in animals using genetic and immunological approaches to improve treatments and diagnostics. In contrast, only few tried to investigate the CyHV-3 replication behaviour in available cell cultures. Whereas, obtaining high virus yields by in vitro replication enables achieving of the mentioned above goals easier and more reliable. The following work presents an attempt to illuminate the KHV replication in common carp brain (CCB) cell cultures from the engineering point of view. The isolate KHV-TP30 was used testing the influence on process parameters, such as multiplicity of infection (MOI), time of infection (TOI) and time of harvest (TOH). Virus concentrations and infectivity at different time points of infection were examined using hydrolyzed probe qPCR (Gilad et al. 2004) and 50% tissue culture infectivity dose (TCID50). The data obtained show that while the amount of the virus DNA remains constant after reaching its maximum, the infectivity of the virus decreases. Thus, especially, TOH can be crucial for generating a high-quality virus stock. Applying optimized parameters improved the infectivity of the harvested virus and reached a robust titre as high as 1.9 × 108 TCID50/mL. To our knowledge, so far, there is no information in the peer-reviewed literature showing comparably high virus titres. Such virus yields not only facilitate conduction of further studies, including stability tests of the virus stock under various supplementation or disinfection trails, but also provide enough virus material to perform more detailed examinations of the infection mechanism.
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References
Aggarwal K, Jing F, Maranga L, Liu J (2011) Bioprocess optimization for cell culture based influenza vaccine production. Vaccine 29(17):3320–3328. doi:10.1016/j.vaccine.2011.01.081
Aoki T, Hirono I, Kurokawa K, Fukuda H, Nahary R, Eldar A et al (2007) Genome sequences of three koi herpesvirus isolates representing the expanding distribution of an emerging disease threatening koi and common carp worldwide. J Virol 81(10):5058–5065. doi:10.1128/JVI.00146-07
Aurelian L (1969) Factors affecting the growth of canine herpesvirus in dog kidney cells. Appl Microbiol 17(1):179–181
Bergmann SM, Kempter J, Sadowski J, Fichtner D (2006) First detection, confirmation and isolation of koi herpesvirus (KHV) in cultured common carp (Cyprinus carpio L.) in Poland. Bull Eur Assoc Fish Pathol 26(2):97–104
Brogden G, Adamek M, Proepsting MJ, Ulrich R, Naim HY, Steinhagen D (2015) Cholesterol-rich lipid rafts play an important role in the cyprinid herpesvirus 3 replication cycle. Vet Microbiol 179(3–4):204–212. doi:10.1016/j.vetmic.2015.05.024
Davidovich M, Dishon A, Ilouze M, Kotler M (2007) Susceptibility of cyprinid cultured cells to cyprinid herpesvirus 3. Arch Virol 152(8):1541–1546. doi:10.1007/s00705-007-0975-4
Dong C, Li X, Weng S, Xie S, He J (2013) Emergence of fatal European genotype CyHV-3/KHV in mainland China. Vet Microbiol 162(1):239–244. doi:10.1016/j.vetmic.2012.10.024
Fuchs W, Fichtner D, Bergmann SM, Mettenleiter TC (2011) Generation and characterization of koi herpesvirus recombinants lacking viral enzymes of nucleotide metabolism. Arch Virol 156(6):1059–1063. doi:10.1007/s00705-011-0953-8
Gilad O, Yun S, Zagmutt-Vergara FJ, Leutenegger CM, Bercovier H, Hedrick RP (2004) Concentrations of a koi herpesvirus (KHV) in tissues of experimentally-infected Cyprinus carpio koi as assessed by real-time TaqMan PCR. Dis Aquat Org 60:179–187. doi:10.3354/dao060179
Hedrick RP, Gilad O, Yun S, Spangenberg JV, Marty GD, Nordhausen RW et al (2000) A herpesvirus associated with mass mortality of juvenile and adult koi, a strain of common carp. J Aquat Anim Health 12(1):44–57. doi:10.1577/1548-8667(2000)012<0044:AHAWMM>2.0.CO;2
Lin S-L, Cheng Y-H, Wen C-M, Chen S-N (2013) Characterization of a novel cell line from the caudal fin of koi carp Cyprinus carpio. J Fish Biol 82(6):1888–1903. doi:10.1111/jfb.12116
Liu Y, Li S (2015) A cell culture model of latent and lytic herpes simplex virus type 1 infection in spiral ganglion. ORL 77(3):141–149. doi:10.1159/000381679
Maranga L, Brazao TF, Carrondo MJT (2003) Virus-like particle production at low multiplicities of infection with the baculovirus insect cell system. Biotechnol Bioeng 84(2):245–253. doi:10.1002/bit.10773
Mendonça RZ, Palomares LA, Ramírez OT (1999) An insight into insect cell metabolism through selective nutrient manipulation. J Biotechnol 72(1–2):61–75. doi:10.1016/S0168-1656(99)00094-2
Mettenleiter TC, Klupp BG, Granzow H (2009) Herpesvirus assembly: an update. Virus Res 143(2):222–234. doi:10.1016/j.virusres.2009.03.018
Michel B, Leroy B, Stalin Raj V, Lieffrig F, Mast J, Wattiez R et al (2010) The genome of cyprinid herpesvirus 3 encodes 40 proteins incorporated in mature virions. J Gen Virol 91(2):452–462. doi:10.1099/vir.0.015198-0
Monaghan SJ, Thompson KD, Adams A, Bergmann SM (2014) Sensitivity of seven PCRs for early detection of koi herpesvirus in experimentally infected carp, Cyprinus carpio L., by lethal and non-lethal sampling methods. J Fish Dis 38(3):303–319. doi:10.1111/jfd.12235
Neukirch M, Böttcher K, Bunnajirakul P (1999) Isolation of a virus from koi with altered gills. Bull Eur Assoc Fish Pathol 19(5):221–224
Nielsen LK, Smyth GK, Greenfield F (1992) Accuracy of the endpoint assay for virus titration. Cytotechnology 8:231–236
Pecson BM, Martin LV, Kohn T (2009) Quantitative PCR for determining the infectivity of bacteriophage MS2 upon inactivation by heat, UV-B radiation, and singlet oxygen: advantages and limitations of an enzymatic treatment to reduce false-positive results. Appl Environ Microbiol 75(17):5544–5554. doi:10.1128/AEM.00425-09
Radford KM, Reid S, Greenfield PF (1997) Substrate limitation in the baculovirus expression vector system. Biotechnol Bioeng 56(1):32–44
Reed LJ, Muench H (1938) A simple method of estimating fifty per cent endpoints. Am J Hyg 27(3):493–497
Ronen A, Perelberg A, Abramowitz J, Hutoran M, Tinman S, Bejerano I, Steinitz M, Kotler M (2003) Efficient vaccine against the virus causing a lethal disease in cultured Cyprinus carpio. Vaccine 21(32):4677–4684. doi:10.1016/S0264-410X(03)00523-1
Timm A, Yin J (2012) Kinetics of virus production from single cells. Virology 424(1):11–17. doi:10.1016/j.virol.2011.12.005
Tu C, Lu Y-P, Hsieh C-Y, Huang S-M, Chang S-K, Chen M-M (2014) Production of monoclonal antibody against ORF72 of koi herpesvirus isolated in Taiwan. Folia Microbiol 59(2):159–165. doi:10.1007/s12223-013-0261-7
Wang Y, Zeng W, Li Y, Liang H, Liu C, Pan H, Lee P, Wu S, Bergmann SM, Wang Q (2015) Development and characterization of a cell line from the snout of koi (Cyprinus carpio L.) for detection of koi herpesvirus. Aquaculture 435:310–317. doi:10.1016/j.aquaculture.2014.10.006
Acknowledgements
This work was supported by the Federal Ministry of Food and Agriculture through the project “Maßnahmen gegen Virosen in der ökologischen Aquakultur” with the support code 2810OE053.
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Editor: Tetsuji Okamoto
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Mletzko, A., Amtmann, A., Bergmann, S. et al. Inoculation of cyprinid herpesvirus 3 (CyHV-3) on common carp brain cells—influence of process parameters on virus yield. In Vitro Cell.Dev.Biol.-Animal 53, 579–585 (2017). https://doi.org/10.1007/s11626-017-0170-1
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DOI: https://doi.org/10.1007/s11626-017-0170-1