Inoculation of cyprinid herpesvirus 3 (CyHV-3) on common carp brain cells—influence of process parameters on virus yield

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 (TCID). 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 × 10 TCID/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.

Virucidal effects of various agents—including protease—against koi herpesvirus and viral haemorrhagic septicaemia virus

“…Virus titres were determined by endpoint titration via TCID 50 assay in 96‐well plates according to Reed and Muench () as previously described by Mletzko et al (). CCB cells were utilized for determination of KHV titres, whereas RT/F cells were used to appoint titres of VHSV.…”

“…In a recent study it was determined that infectivity of KHV during in vitro replication decreases after reaching the maximum titre with progressing cell lysis (Mletzko et al, ). This could be due to proteolytic enzymes released from the cells that affect the proteins in the viral envelope, which in consequence perturbs virus entry into the cells and thus viral replication.…”

Genomic and biologic comparisons of cyprinid herpesvirus 3 strains

“…The Cavoy strain was amplified from a commercial aliquot of an attenuated vaccine produced by passage in cell culture of an isolate from Israel [ 19 – 21 ]. The T strain was subcloned from a large viral plaque obtained after 30 passages of a Taiwanese isolate on a koi fin cell line [ 22 ]. GZ11-SC is a subclone of the GZ11 strain from China [ 6 ].…”

Current knowledge and future prospects of vaccines against cyprinid herpesvirus 3 (CyHV-3)

“…Of note, a negative correlation was observed between the viral growth in vitro and the virulence in vivo inferring that, in general, the more adapted a viral strain is to cell culture, the more attenuated it is in vivo. This was the case for the highly cell cultureadapted CyHV-3 T strain [13]. The apparent low genomic diversity found among CyHV-3 strains can be considered as an advantage in terms of vaccine development.…”

“…Similarly, the ORF123 dUTPase deletion is associated with partial attenuation. Recently, using an eukaryotic recombination based approach, a double deletion of both ORF55 TK and ORF123 dUTPase has been described in two independent viral strains (CyHV-3 I, low cell culture adaptation/high virulence profile [45]; and CyHV-3 T, high cell culture adaptation/low virulence profile [11,13]) [46]. In both cases, an important attenuation was observed, and significant protection was ensured following a lethal challenge with the parental strain (Table 2) [46].…”