title: Virus replication cycle of white spot syndrome virus in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei.
abstract: The replication cycle of white spot syndrome virus (WSSV) was investigated in secondary cell cultures from the lymphoid organ of Litopenaeus vannamei. The secondary cells formed a confluent monolayer at 24 h post-reseeding, and this monolayer could be maintained for 10 days with a viability of 90 %. Binding of WSSV to cells reached a maximum (73 ± 3 % of cells and 4.84 ± 0.2 virus particles per virus-binding cell) at 120 min at 4 °C. WSSV entered cells by endocytosis. The co-localization of WSSV and early endosomes was observed starting from 30 min post-inoculation (p.i.). Double indirect immunofluorescence staining showed that all cell-bound WSSV particles entered these cells in the period between 0 and 60 min p.i. and that the uncoating of WSSV occurred in the same period. After 1 h inoculation at 27 °C, the WSSV nucleocapsid protein VP664 and envelope protein VP28 started to be synthesized in the cytoplasm from 1 and 3 h p.i., and were transported into nuclei from 3 and 6 h p.i., respectively. The percentage of cells that were VP664- and VP28-positive in their nuclei peaked (50 ± 4 %) at 12 h p.i. Quantitative PCR showed that WSSV DNA started to be synthesized from 6 h p.i. In vivo titration of the supernatants showed that the progeny WSSV were released from 12 h p.i. and peaked at 18 h p.i. In conclusion, the secondary cell cultures from the lymphoid organ were proven to be ideal for examination of the replication cycle of WSSV.
authors.affiliation: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Wenfeng
authors.initials: W
authors.lastname: Li
authors.affiliation: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Lowiese M B
authors.initials: LMB
authors.lastname: Desmarets
authors.affiliation: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Gaëtan M A
authors.initials: GMA
authors.lastname: De Gryse
authors.affiliation: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Sebastiaan
authors.initials: S
authors.lastname: Theuns
authors.affiliation: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Vo
authors.initials: V
authors.lastname: Van Tuan
authors.affiliation: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Khuong
authors.initials: K
authors.lastname: Van Thuong
authors.affiliation: Laboratory of Aquaculture & Artemia Reference Center, Department of Animal Production, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium.
authors.forename: Peter
authors.initials: P
authors.lastname: Bossier
authors.affiliation: Laboratory of Virology, Department of Virology, Parasitology and Immunology, Faculty of Veterinary Medicine, Ghent University, Ghent, Belgium.
authors.forename: Hans J
authors.initials: HJ
authors.lastname: Nauwynck
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods*; D002467:Cell Nucleus / Q000821:virology; D008221:Lymphoid Tissue / Q000821:virology; D019590:Nucleocapsid Proteins / Q000235:genetics / Q000378:metabolism; D033561:Penaeidae / Q000821:virology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism; D014779:Virus Replication; D046848:White spot syndrome virus 1 / Q000235:genetics / Q000502:physiology*
chemical_list: D019590:Nucleocapsid Proteins; D014759:Viral Envelope Proteins